Methods of treating muscular dystrophy

ABSTRACT

Disclosed herein are α7β1 integrin modulatory agents and methods of using such to treat conditions associated with decreased α7β1 integrin expression or activity, including muscular dystrophy. In one example, methods for treating a subject with muscular dystrophy are disclosed. The methods include administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy. Also disclosed are methods of enhancing muscle regeneration, repair, or maintenance in a subject and methods of enhancing α7β1 integrin expression by use of the disclosed α7β1 integrin modulatory agents. Methods of prospectively preventing or reducing muscle injury or damage in a subject are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.14/776,898 which is the U.S. National Stage of International ApplicationNo. PCT/US2014/029085, filed Mar. 14, 2014, which was published inEnglish under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Patent Application No. 61/798,479, filed on Mar. 15,2013, each of which is herein incorporated by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Numbers R43AR060030, R21 NS058429-01, and R21 AR060769 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD

This disclosure relates to the field of muscular dystrophy and inparticular, to compositions and methods for treating muscular dystrophy,such as Duchenne muscular dystrophy, Fukuyama congenital musculardystrophy or merosin deficient congenital muscular dystrophy type 1A or1D.

BACKGROUND

Mutations in the α7 integrin gene are responsible for congenitalmyopathy in man. The α7β1 integrin is also a major modifier of muscledisease progression in various genetic muscle diseases including varioustypes of muscular dystrophy, such as Duchenne muscular dystrophy (DMD),Fukuyama congenital muscular dystrophy (FCMD) and merosin deficientcongenital muscular dystrophy type 1A (MDC1A). However, transcriptionalregulation of the α7 integrin gene, including such role in musculardystrophy (e.g., DMD, FCMD and/or MDC1A), remains poorly understood.

Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disease andthe most common form of muscular dystrophy. DMD affects 1 in 3500 livemale births with patients suffering from chronic muscle degeneration andweakness. Clinical symptoms are first detected between the ages of 2 and5 years and, by the time the patient is in their teens, the ability forindependent ambulation is lost. Death typically occurs in the patientbefore they are 30 years old due to cardiopulmonary failure.

Fukuyama congenital muscular dystrophy (FCMD) and MDC1A are congenitalmuscular dystrophies that are heritable neuromuscular disorders. MDC1Ais characterized by muscle weakness at birth or in infancy Affectedinfants will present with poor muscle tone and few movements. Thequality of life and life span of the child is affected throughprogressive muscle wasting, respiratory compromise, and spinal rigidity.MDC1A is the most common and severe form of congenital musculardystrophy, accounting for 30-40% of all congenital muscular dystrophy(CMD) diagnosed cases. MDC1A is characterized by congenital hypotonia,distinct joint contractures, and a lack of independent ambulation.Feeding tube placement and positive pressure ventilation is oftenrequired for the respiratory problems that occur. Patients afflictedwith MDC1A often die before they reach the age of ten years. FCMD iscaused by mutations in the fukutin gene, located at human chromosome9q31. The disease is inherited in an autosomal recessive manner. FCMD isa type of Limb-Girdle muscular dystrophy. Currently there is no cure forDMD, FCMD or MDC1A.

SUMMARY

The muscular dystrophies are a group of diverse, heritable neuromusculardisorders which represent a group of devastating neuromuscular diseasescharacterized by primary or secondary skeletal muscle involvement.Currently, there are no cures for such diseases.

Disclosed herein are α7β1 integrin expression modulatory agents andmethods of using such to treat a condition associated with impaired α7integrin expression, such as muscular dystrophy. In one embodiment, amethod for treating a subject with muscular dystrophy is disclosed. Themethod includes administering an effective amount of an α7β1 integrinmodulatory agent to the subject with muscular dystrophy, wherein theα7β1 integrin modulatory agent is a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18, or acombination thereof, wherein the α7β1 integrin modulatory agentincreases α7β1 integrin expression or activity as compared to α7β1integrin expression or activity prior to treatment, thereby treating thesubject with muscular dystrophy (such as MDC1A, MDC1D, LGMD, DMD, FCMDor FHMD).

Also disclosed are methods of enhancing muscle regeneration, repair, ormaintenance in a subject. In some embodiments, the method includesadministering an effective amount of an α7β1 integrin modulatory agentto the subject in need of muscle regeneration, repair or maintenance,wherein the α7β1 integrin modulatory agent comprises a compoundencompassed by any one of Formulas 1-16, or provided by any one ofTables 1-16 and 18, or a combination thereof, wherein the α7β1 integrinmodulatory agent increases α7β1 integrin expression or activity ascompared to α7β1 integrin expression or activity prior to treatment,thereby enhancing muscle regeneration, repair or maintenance in asubject. In a specific embodiment, the present disclosure provides amethod for increasing muscle regeneration in a subject. For example,geriatric subjects, subjects suffering from muscle disorders, andsubjects suffering from muscle injury, including activity induced muscleinjury, such as injury caused by exercise, may benefit from thisembodiment.

In yet further embodiments of the disclosed method, the α7β1 integrinmodulatory agent is administered in a preventative manner, such as toprevent or reduce muscular damage or injury (such as activity orexercise induced injury). For example, geriatric subjects, subjectsprone to muscle damage, or subjects at risk for muscular injury, such asathletes, may be treated in order to eliminate or ameliorate musculardamage, injury, or disease.

Further disclosed are methods of enhancing α7β1 integrin expression. Insome embodiments, the method includes contacting a cell with aneffective amount of an α7β1 integrin modulatory agent, wherein the α7β1integrin modulatory agent includes a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18, or acombination thereof and increases α7β1 integrin expression in thetreated cell relative to α7β1 integrin expression in an untreated cell,thereby enhancing α7β1 integrin expression. The methods of the presentdisclosure can include administering the α7β1 integrin modulatory agentwith one or more additional pharmacological substances, such as atherapeutic agent. In some aspects, the additional therapeutic agentenhances the therapeutic effect of the α7β1 integrin modulatory agent.In further aspects, the therapeutic agent provides independenttherapeutic benefit for the condition being treated. In variousexamples, the additional therapeutic agent is a component of theextracellular matrix, such as an integrin, dystrophin, dystroglycan,utrophin, or a growth factor. In further examples, the therapeutic agentreduces or enhances expression of a substance that enhances theformation or maintenance of the extracellular matrix. In some examples,the therapeutic agent is an additional α7β1 integrin modulatory agentsuch as laminin-111, a laminin-111 fragment, valproic acid or a valproicacid analog.

In some examples, the α7β1 integrin modulatory agent is applied to aparticular area of the subject to be treated. For example, the α7β1integrin modulatory agent may be injected into a particular area to betreated, such as a muscle. In further examples, the α7β1 integrinmodulatory agent is administered such that it is distributed to multipleareas of the subject, such as systemic administration or regionaladministration.

A α7β1 integrin modulatory agent, can be administered by any suitablemethod, such as topically, parenterally (such as intravenously orintraperitoneally), or orally. In a specific example, the α7β1 integrinmodulatory agent is administered systemically, such as throughparenteral administration, such as stomach injection or peritonealinjection.

Although the disclosed methods generally have been described withrespect to muscle regeneration, the disclosed methods also may be usedto enhance repair or maintenance, or prevent damage to, other tissuesand organs. For example, the methods of the present disclosure can beused to treat symptoms of muscular dystrophy stemming from effects tocells or tissue other than skeletal muscle, such as impaired or alteredbrain function, smooth muscles, or cardiac muscles.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a digital image illustrating the results of quantitativereal-time PCR used to assess Itga7, Itgb1, and Lama2 transcript levelsin C2C12 myoblasts and myotubes treated for 24 hours with DMSO control,10 μM MLS000683232-01 (IED-232), 10 μM MLS001165937-01 (IED-937),Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 inHPBCD. * denotes a significant difference in relative transcript levelswith ** p-value<0.01 and *** p<0.001.

FIG. 2 is a digital image of Western Blots and quantitative analysis ofα7 Integrin and GAPDH protein levels in C2C12 myotubes treated for 48hours with DMSO control, 10 μM MLS000683232-01 (IED-232),Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 inHPBCD. Bands were quantified using Image J software and then graphed asα7 Integrin protein levels relative to GAPDH protein levels. * denotes asignificant difference in relative protein levels with ** p<0.01.

FIG. 3 is an image of results (fluorescence relative to DMSO at variousconcentrations of the agent) from a screen using particular embodimentsof the disclosed α7β1 integrin modulatory agents.

FIG. 4A is an image of the relative fluorescence generated by variousconcentrations of beta-galactosidase from the FDG substrate (FIG. 4A).The effects of DMSO concentrations on α7^(+/LacZ) myoblasts (FIG. 4B)and myotube (FIG. 4C) assays were determined to be unacceptably high atconcentrations greater than 1%. (FIG. 4D) The α7^(+/LacZ) myotube assayresponse with varying levels of FDG in the FDG Buffer.

FIGS. 5A and 5B are graphs illustrating dose-response curves for SU9516on α7^(+/LacZ) myoblasts (FIG. 5A) or myotubes (FIG. 5B). Thenon-conventional appearance of the curves is thought to be a mechanismof concentration dependent cellular toxicity.

FIG. 6 is an image of a table providing curve classification resultsfrom a primary myoblast screen disclosed herein.

FIGS. 7A-7F provide graphs of myotube dose-response and thecorresponding chemical structures [SU9516 (FIG. 7A), MLS000532969 (FIG.7B), MLS003126425 (FIG. 7C), MLS001060533 (FIG. 7D), MLS000683232 (FIG.7E), and MLS000683234 (FIG. 7F)] for each compound examined whereinn=6-9 over multiple days for all points.

FIGS. 8A and 8B provide results obtained for C2C12 myotubes treated withDMSO (n=3) or 12 μM SU9516 (n=3), which were examined for α7 Integrinprotein levels by western blotting (FIG. 8A) wherein GapDH proteinlevels were used as a loading normalization control and the α7Integrin/GapDH levels were quantitated and graphed for both DMSO andSU9516 treatments (FIG. 8B).

FIG. 9 is an image of results obtained for various different compoundsdisclosed herein and their effects on α7 integrin levels in human DMDmyotubes.

FIG. 10 is an image illustrating chemical structures and names,similarity score relative to Stryka-969, and ITGA7^(+/LacZ) myotubedose-response for 4 compounds with similar structures to Stryka-969.

FIG. 11 is an image of a Western blot analysis for α7 Integrin andα-tubulin, quantitated and graphed wherein significance was determinedby unpaired t-test with ** p<0.01 and *** p<0.001.

FIG. 12 provides an exemplary synthetic scheme for making specific α7β1integrin modulatory agents disclosed herein.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Overview of SeveralEmbodiments

Disclosed herein are α7β1 integrin expression modulatory agents andmethods of using such to treat a condition associated with impaired α7integrin expression, such as muscular dystrophy.

In one embodiment, a method for treating a subject with musculardystrophy is disclosed. The method includes administering an effectiveamount of an α7β1 integrin modulatory agent to the subject with musculardystrophy, wherein the α7β1 integrin modulatory agent is a compoundencompassed by any one of Formulas 1-16, or provided by any one ofTables 1-16 and 18, or a combination thereof, wherein the α7β1 integrinmodulatory agent increases α7β1 integrin expression or activity ascompared to α7β1 integrin expression or activity prior to treatment,thereby treating the subject with muscular dystrophy.

In some embodiments, a method for treating a subject with musculardystrophy, comprises administering an effective amount of an α7β1integrin modulatory agent to the subject with muscular dystrophy,wherein the α7β1 integrin modulatory agent comprises a compound having aformula selected from any one of the following

wherein R¹, R^(1′), R², and R³ each independently is as specified inTable 3, Table 4, Table 5, and/or Table 6. In some embodiments, acombination of any of these compounds, or any other α7β1 integrinmodulatory agents disclosed herein may be used. The α7β1 integrinmodulatory agent increases α7β1 integrin expression or activity ascompared to α7β1 integrin expression or activity prior to treatment,thereby treating the subject with muscular dystrophy. In someembodiments, the compound may be selected from any of those provided inany one of Tables 1-16 and 18.

In some embodiments, the muscular dystrophy is merosin deficientcongenital muscular dystrophy Type 1A (MDC1A), merosin deficientcongenital muscular dystrophy Type 1D (MDC1D), limb-girdle musculardystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenitalmuscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy(FHMD).

In some particular embodiments, the muscular dystrophy is DMD, MDC1A orFCMD.

In one particular embodiment, the muscular dystrophy is DMD.

In some embodiments, the α7β1 integrin modulatory agent is administeredwith an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a costamericprotein, a growth factor, satellite cells, stem cells, myocytes or anadditional α7β1 integrin modulatory agent.

In some embodiments, the additional α7β1 integrin modulatory agent islaminin-111, a laminin-111 fragment, valproic acid, or a valproic acidanalog.

In some embodiments, the method further includes selecting a subjectwith muscular dystrophy.

In some embodiments, the selecting a subject with muscular dystrophyincludes diagnosing the subject with muscular dystrophy prior toadministering an effective amount of the α7β1 integrin modulatory agentto the subject.

In other embodiments, a method of enhancing muscle regeneration, repair,or maintenance in a subject is disclosed.

In some embodiments, the method includes administering an effectiveamount of an α7β1 integrin modulatory agent to the subject with musculardystrophy, wherein the α7β1 integrin modulatory agent comprises acompound encompassed by any one of Formulas 1-16, or provided by any oneof Tables 1-16 and 18, or a combination thereof, wherein the α7β1integrin modulatory agent increases α7β1 integrin expression or activityas compared to α7β1 integrin expression or activity prior to treatment,thereby enhancing muscle regeneration, repair or maintenance in asubject.

In some embodiments, the method includes administering the α7β1modulatory agent prior to the subject experiencing muscle damage ordisease.

In some embodiments, the method is a method of enhancing musclemaintenance in a subject.

In some embodiments, the α7β1 integrin modulatory agent is administeredto the subject prior to the subject exercising.

In some embodiments, the α7β1 integrin modulatory agent is administeredto a subject at risk of acquiring a muscle disease or damage, such as anelderly subject.

In some embodiments, the method also includes selecting a subject inneed of enhancing muscle regeneration, repair, or maintenance.

In some embodiments, selecting a subject in need of enhancing muscleregeneration, repair, or maintenance includes diagnosing the subjectwith a condition characterized by impaired muscle regeneration prior toadministering an effective amount of the α7β1 integrin modulatory agentto the subject.

In some embodiments, selecting a subject in need of enhancing muscleregeneration, repair, or maintenance comprises diagnosing the subjectwith a condition characterized by impaired production of a component ofα7β1 integrin prior to administering an effective amount of the α7β1integrin modulatory agent to the subject.

In some embodiments, the α7β1 integrin modulatory agent is administeredwith an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a costamericprotein, a growth factor, satellite cells, stem cells, myocytes or anadditional α7β1 integrin modulatory agent.

In some embodiments, the additional α7β1 integrin modulatory agent islaminin-111, a laminin-111 fragment, valproic acid, or a valproic acidanalog.

In further embodiments, a method of prospectively preventing or reducingmuscle injury or damage in a subject is disclosed.

In some embodiments, the method includes administering an effectiveamount of an α7β1 integrin modulatory agent to the subject wherein theα7β1 integrin modulatory agent includes a compound encompassed by anyone of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or acombination thereof, wherein the α7β1 integrin modulatory agentincreases α7β1 integrin expression or activity as compared to α7β1integrin expression or activity prior to treatment, therebyprospectively preventing or reducing muscle injury or damage in thesubject.

In some embodiments, the subject is at risk of developing a muscleinjury or damage.

In some embodiments, the α7β1 integrin modulatory agent is administeredwith an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a costamericprotein, a growth factor, satellite cells, stem cells, myocytes or anadditional α7β1 integrin modulatory agent.

In some embodiments, the additional α7β1 integrin modulatory agent islaminin-111, a laminin-111 fragment, valproic acid, or a valproic acidanalog.

In even further embodiments, a method of enhancing α7β1 integrinexpression is provided.

In some embodiments, the method includes contacting a cell with aneffective amount of an α7β1 integrin modulatory agent, wherein the α7β1integrin modulatory agent includes a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18, or acombination thereof and increases α7β1 integrin expression in thetreated cell relative to α7β1 integrin expression in an untreated cell,thereby enhancing α7β1 integrin expression.

In some embodiments, the cell is a muscle cell.

In some embodiments, the muscle cell is present in a mammal, and whereincontacting the cell with an agent comprises administering the agent tothe mammal.

II. Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. The term“comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology may be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment.

A person of ordinary skill in the art would recognize that the abovedefinitions are not intended to include impermissible substitutionpatterns (e.g., methyl substituted with 5 different groups, pentavalentcarbon, and the like). Such impermissible substitution patterns areeasily recognized by a person of ordinary skill in the art.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Allsequences provided in the disclosed Genbank Accession numbers areincorporated herein by reference as available on Aug. 11, 2011. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject one or more agents, such asan agent that increases α7β1 expression and/or treats one or moresymptoms associated with muscular dystrophy, by any effective route.Exemplary routes of administration include, but are not limited to,injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, and intravenous), oral, sublingual, rectal,transdermal, intranasal, vaginal and inhalation routes.

Agent: Any protein, nucleic acid molecule (including chemically modifiednucleic acids), compound, antibody, small molecule, organic compound,inorganic compound, or other molecule of interest. Agent can include atherapeutic agent, a diagnostic agent or a pharmaceutical agent. Atherapeutic or pharmaceutical agent is one that alone or together withan additional compound induces the desired response (such as inducing atherapeutic or prophylactic effect when administered to a subject,including treating a subject with a muscular dystrophy).

In some examples, an agent can act directly or indirectly to alter theexpression and/or activity of α7β1. In a particular example, atherapeutic agent significantly increases the expression and/or activityof α7β1 (which is a muscular dystrophy associated molecule) therebytreating one or more signs or symptoms associated with musculardystrophy. An example of a therapeutic agent is one that can increasethe expression and/or activity of the α7β1 gene or gene product, forexample as measured by a clinical response (such as a decrease in one ormore signs or symptoms associated with the muscular dystrophy, animprovement in muscular health, regeneration, repair or maintenance of amuscle cell or tissue). “Improving muscular health” refers to animprovement in muscular health compared with a preexisting state orcompared with a state which would occur in the absence of treatment. Forexample, improving muscular health may include enhancing muscleregeneration, maintenance, or repair. Improving muscular health may alsoinclude prospectively treating a subject to prevent or reduce musculardamage or injury. “Regeneration” refers to the repair of cells ortissue, such as muscle cells or tissue (or organs) which includes musclecells, following injury or damage to at least partially restore themuscle or tissue to a condition similar to which the cells or tissueexisted before the injury or damage occurred. Regeneration also refersto facilitating repair of cells or tissue in a subject having a diseaseaffecting such cells or tissue to eliminate or ameliorate the effects ofthe disease. In more specific examples, regeneration places the cells ortissue in the same condition or an improved physiological condition asbefore the injury or damage occurred or the condition which would existin the absence of disease. “Maintenance” of cells or tissue, such asmuscle cells or tissue (or organs) which includes muscle cells, refersto maintaining the cells or tissue in at least substantially the samephysiological condition, such as maintaining such condition even in thepresence of stimulus which would normally cause damage, injury, ordisease. “Repair” of cells or tissue, such as muscle cells or tissue (ororgans) which includes muscle cells, refers to the physiological processof healing damage to the cells or tissue following damage or othertrauma.

A “pharmaceutical agent” is a chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect whenadministered to a subject, alone or in combination with anothertherapeutic agent(s) or pharmaceutically acceptable carriers. In aparticular example, a pharmaceutical agent significantly increases theexpression and/or activity of α7β1 thereby treating a condition ordisease associated with decreased α7β1 expression/activity, such asmuscular dystrophy.

Acyl: H—C(O)—, alkyl-C(O)—, substituted alkyl-C(O)—, cycloalkyl-C(O)—,substituted cycloalkyl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O)—, heterocyclyl-C(O)—, and substitutedheterocyclyl-C(O)—.

Acylamino: —NR^(a)C(O)alkyl, —NR^(a)C(O) substituted alkyl,—NR^(a)C(O)cycloalkyl, —NR^(a)C(O) substituted cycloalkyl,—NR^(a)C(O)cycloalkenyl, —NR^(a)C(O) substituted cycloalkenyl,—NR^(a)C(O)alkenyl, —NR^(a)C(O) substituted alkenyl, —NR^(a)C(O)alkynyl,—NR^(a)C(O) substituted alkynyl, —NR^(a)C(O)aryl, —NR^(a)C(O)substituted aryl, —NR^(a)C(O)heteroaryl, —NR^(a)C(O) substitutedheteroaryl, —NR^(a)C(O)heterocyclyl, and —NR^(a)C(O) substitutedheterocyclyl, wherein R^(a) is selected from hydrogen, alkyl, aryl, andcycloalkyl.

Acyloxy: alkyl-C(O)O—, substituted alkyl-C(O)O—, aryl-C(O)O—,substituted aryl-C(O)O—, cycloalkyl-C(O)O—, substitutedcycloalkyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—,heterocyclyl-C(O)O—, and substituted heterocyclyl-C(O)O—.

Acylalkyloxy: alkyl-C(O)alkylO—, substituted alkyl-C(O)alkylO—,aryl-C(O)alkylO—, substituted aryl-C(O)alkylO—, cycloalkyl-C(O)alkylO—,substituted cycloalkyl-C(O)alkylO—, heteroaryl-C(O)alkylO—, substitutedheteroaryl-C(O)alkylO—, heterocyclyl-C(O)alkylO—, and substitutedheterocyclyl-C(O)alkylO—.

Alkyl: A saturated or unsaturated monovalent hydrocarbon having a numberof carbon atoms ranging from one to ten (e.g., C₁₋₁₀alkyl), which isderived from removing one hydrogen atom from one carbon atom of a parentcompound (e.g., alkane, alkene, alkyne). An alkyl group may be branchedor straight-chain.

Alkenyl: A unsaturated monovalent hydrocarbon having a number of carbonatoms ranging from one to ten (e.g., C₂₋₁₀alkenyl), which has at leastone carbon-carbon double bond and is derived from removing one hydrogenatom from one carbon atom of a parent alkene. An alkenyl group may bebranched, straight-chain, cyclic, cis, or trans (e.g., E or Z).

Alkynyl: A unsaturated monovalent hydrocarbon having a number of carbonatoms ranging from one to ten (e.g., C₂₋₁₀ alkynyl), which has at leastone carbon-carbon triple bond and is derived from removing one hydrogenatom from one carbon atom of a parent alkyne. An alkynyl group may bebranched, straight-chain, or cyclic.

Alkoxy: —O-alkyl (e.g., methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, t-butoxy, sec-butoxy, n-pentoxy).

Alkylthio: —S-alkyl, wherein alkyl is as defined herein. This term alsoencompasses oxidized forms of sulfur, such as —S(O)-alkyl, or—S(O)₂-alkyl.

Amino: —NH₂.

Aminocarbonyl: —C(O)N(R^(b))₂, wherein each R^(b) independently isselected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) mayoptionally be joined together with the nitrogen bound thereto to form aheterocyclyl or substituted heterocyclyl group, provided that both R^(b)are not both hydrogen.

Aminocarbonylalkyl: -alkylC(O)N(R^(b))₂, wherein each R^(b)independently is selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also,each R^(b) may optionally be joined together with the nitrogen boundthereto to form a heterocyclyl or substituted heterocyclyl group,provided that both R^(b) are not both hydrogen.

Aminocarbonylamino: —NR^(a)C(O)N(R^(b))₂, wherein R^(a) and each R^(b)are as defined herein.

Aminodicarbonylamino: —NR^(a)C(O)C(O)N(R^(b))₂, wherein R^(a) and eachR^(b) are as defined herein.

Aminocarbonyloxy: —O—C(O)N(R^(b))₂, wherein each R^(b) independently isas defined herein.

Aminosulfonyl: —SO₂N(R^(b))₂, wherein each R^(b) independently is asdefined herein.

Analog or Derivative: A compound which is sufficiently homologous to acompound such that it has a similar functional activity for a desiredpurpose as the original compound. Analogs or derivatives refers to aform of a substance, such as cholestan, which has at least onefunctional group altered, added, or removed, compared with the parentcompound. In some examples, examples of an analog are provided in Tables1-6, for example. “Functional group” refers to a radical, other than ahydrocarbon radical, that adds a physical or chemical property to asubstance.

Aryl: a monovalent aromatic carbocyclic group of from 6 to 15 carbonatoms having a single ring (e.g., phenyl) or multiple condensed rings(e.g., naphthyl), which condensed rings may or may not be aromaticprovided that the point of attachment is through an atom of the aromaticaryl group.

Aryloxy —O-aryl.

Arylthio —S-aryl, wherein aryl is as defined herein. This term alsoencompasses oxidized forms of sulfur, such as —S(O)-aryl, or—S(O)₂-aryl.

Biological activity: The beneficial or adverse effects of an agent onliving matter. When the agent is a complex chemical mixture, thisactivity is exerted by the substance's active ingredient orpharmacophore, but can be modified by the other constituents. Activityis generally dosage-dependent and it is not uncommon to have effectsranging from beneficial to adverse for one substance when going from lowto high doses. In one example, the agent significantly increases thebiological activity of α7β1 which reduces one or more signs or symptomsassociated with the muscular dystrophy.

Contacting: Placement in direct physical association, including both asolid and liquid form. Contacting an agent with a cell can occur invitro by adding the agent to isolated cells or in vivo by administeringthe agent to a subject.

Control: A sample or standard used for comparison with a test sample,such as a biological sample obtained from a patient (or plurality ofpatients) without a particular disease or condition, such as a musculardystrophy. In some embodiments, the control is a sample obtained from ahealthy patient (or plurality of patients) (also referred to herein as a“normal” control), such as a normal biological sample. In someembodiments, the control is a historical control or standard value(e.g., a previously tested control sample or group of samples thatrepresent baseline or normal values (e.g., expression values), such asbaseline or normal values of a particular gene such as a α7β1 gene, geneproduct in a subject without a muscular dystrophy). In some examples,the control is a standard value representing the average value (oraverage range of values) obtained from a plurality of patient samples(such as an average value or range of values of the gene or geneproducts, such as the α7β1 gene or gene products, in the subjectswithout a muscular dystrophy).

Carboxyl: —COOH or salts thereof.

Carboxyester: —C(O)O-alkyl, —C(O)O— substituted alkyl, —C(O)O-aryl,—C(O)O-substituted aryl, —C(O)O-cycloalkyl, —C(O)O-substitutedcycloalkyl, —C(O)O-heteroaryl, —C(O)O-substituted heteroaryl,—C(O)O-heterocyclyl, and —C(O)O-substituted heterocyclyl.

(Carboxyester)amino: —NR^(a)—C(O)O-alkyl, —NR^(a)—C(O)O— substitutedalkyl, —NR^(a)—C(O)O-aryl, —NR^(a)—C(O)O-substituted aryl,—NR^(a)—C(O)O-cycloalkyl, —NR^(a)—C(O)O-substituted cycloalkyl,—NR^(a)—C(O)O-heteroaryl, —NR^(a)—C(O)O-substituted heteroaryl,—NR^(a)—C(O)O-heterocyclyl, and —NR^(a)—C(O)O-substituted heterocyclyl,wherein R^(a) is as recited herein.

(Carboxyester)oxy: —O—C(O)O-alkyl, —O—C(O)O— substituted alkyl,—O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl,—O—C(O)O-substituted cycloalkyl, —O—C(O)O-heteroaryl,—O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclyl, and—O—C(O)O-substituted heterocyclyl.

Cyano: —CN.

Cycloalkyl: cyclic alkyl (or alkenyl, or alkynyl) groups of from 3 to 10carbon atoms having single or multiple cyclic rings including fused,bridged, and spiro ring systems (e.g., cyclopropyl, cyclobutyl, etc.).

(Cycloalkyl)oxy: —O-cycloalkyl.

(Cycloalkyl)thio: —S-cycloalkyl. This term also encompasses oxidizedforms of sulfur, such as —S(O)— cycloalkyl, or —S(O)₂-cycloalkyl.

Decrease: To reduce the quality, amount, or strength of something. Inone example, a therapy decreases one or more symptoms associated withthe muscular dystrophy, for example as compared to the response in theabsence of the therapy.

Diagnosis: The process of identifying a disease, such as musculardystrophy, by its signs, symptoms and results of various tests. Theconclusion reached through that process is also called “a diagnosis.”Forms of testing commonly performed include blood tests, medicalimaging, urinalysis, and biopsy.

Effective amount: An amount of agent that is sufficient to generate adesired response, such as reducing or inhibiting one or more signs orsymptoms associated with a condition or disease. When administered to asubject, a dosage will generally be used that will achieve targettissue/cell concentrations. In some examples, an “effective amount” isone that treats one or more symptoms and/or underlying causes of any ofa disorder or disease. In some examples, an “effective amount” is atherapeutically effective amount in which the agent alone with anadditional therapeutic agent(s) (for example anti-pathogenic agents),induces the desired response such as treatment of a muscular dystrophy,such as DMD, FCMD or MDC1A.

In particular examples, it is an amount of an agent capable ofincreasing α7β1 gene expression or activity by least 20%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or even at least 100% (elimination of the disease to a pointbeyond detection).

In some examples, an effective amount is an amount of a pharmaceuticalpreparation that alone, or together with a pharmaceutically acceptablecarrier or one or more additional therapeutic agents, induces thedesired response.

In one example, a desired response is to increase the subject's survivaltime by slowing the progression of the disease, such as slowing theprogression of muscular dystrophy. The disease does not need to becompletely inhibited for the pharmaceutical preparation to be effective.For example, a pharmaceutical preparation can decrease the progressionof the disease by a desired amount, for example by at least 20%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or even at least 100%, as compared to theprogression typical in the absence of the pharmaceutical preparation.

In another or additional example, it is an amount sufficient topartially or completely alleviate symptoms of the muscular dystrophywithin the subject. Treatment can involve only slowing the progressionof the disease temporarily, but can also include halting or reversingthe progression of the disease permanently.

Effective amounts of the agents described herein can be determined inmany different ways, such as assaying for a reduction in of one or moresigns or symptoms associated with the muscular dystrophy in the subjector measuring the expression level of one or more molecules known to beassociated with the muscular dystrophy. Effective amounts also can bedetermined through various in vitro, in vivo or in situ assays,including the assays described herein.

The disclosed therapeutic agents can be administered in a single dose,or in several doses, for example daily, during a course of treatment.However, the effective amount can be dependent on the source applied(for example a nucleic acid molecule isolated from a cellular extractversus a chemically synthesized and purified nucleic acid), the subjectbeing treated, the severity and type of the condition being treated, andthe manner of administration.

Expression: The process by which the coded information of a gene isconverted into an operational, non-operational, or structural part of acell, such as the synthesis of a protein. Gene expression can beinfluenced by external signals. For instance, exposure of a cell to ahormone may stimulate expression of a hormone-induced gene. Differenttypes of cells can respond differently to an identical signal.Expression of a gene also can be regulated anywhere in the pathway fromDNA to RNA to protein. Regulation can include controls on transcription,translation, RNA transport and processing, degradation of intermediarymolecules such as mRNA, or through activation, inactivation,compartmentalization or degradation of specific protein molecules afterthey are produced. In an example, expression, such as expression ofα7β1, can be regulated to treat one or more signs or symptoms associatedwith muscular dystrophy.

The expression of a nucleic acid molecule can be altered relative to anormal (wild type) nucleic acid molecule. Alterations in geneexpression, such as differential expression, include but are not limitedto: (1) overexpression; (2) underexpression; or (3) suppression ofexpression. Alternations in the expression of a nucleic acid moleculecan be associated with, and in fact cause, a change in expression of thecorresponding protein.

Protein expression can also be altered in some manner to be differentfrom the expression of the protein in a normal (wild type) situation.This includes but is not necessarily limited to: (1) a mutation in theprotein such that one or more of the amino acid residues is different;(2) a short deletion or addition of one or a few (such as no more than10-20) amino acid residues to the sequence of the protein; (3) a longerdeletion or addition of amino acid residues (such as at least 20residues), such that an entire protein domain or sub-domain is removedor added; (4) expression of an increased amount of the protein comparedto a control or standard amount; (5) expression of a decreased amount ofthe protein compared to a control or standard amount; (6) alteration ofthe subcellular localization or targeting of the protein; (7) alterationof the temporally regulated expression of the protein (such that theprotein is expressed when it normally would not be, or alternatively isnot expressed when it normally would be); (8) alteration in stability ofa protein through increased longevity in the time that the proteinremains localized in a cell; and (9) alteration of the localized (suchas organ or tissue specific or subcellular localization) expression ofthe protein (such that the protein is not expressed where it wouldnormally be expressed or is expressed where it normally would not beexpressed), each compared to a control or standard. Controls orstandards for comparison to a sample, for the determination ofdifferential expression, include samples believed to be normal (in thatthey are not altered for the desired characteristic, for example asample from a subject who does not have muscular dystrophy, such as DMD,FCMD or MDC1A) as well as laboratory values (e.g., range of values),even though possibly arbitrarily set, keeping in mind that such valuescan vary from laboratory to laboratory.

Laboratory standards and values can be set based on a known ordetermined population value and can be supplied in the format of a graphor table that permits comparison of measured, experimentally determinedvalues.

Extracellular matrix: An extracellular structure of a tissue or a layerthereof, including the arrangement, composition, and forms of one ormore matrix components, such as proteins, including structural proteinssuch as collagen and elastin, proteins such as fibronectin and laminins,and proteoglycans. The matrix may comprise fibrillic collagen, having anetwork of fibers. In some examples, the extracellular matrix isconnected to cells through the costameric protein network.

Halogen or Halo: fluoro, chloro, bromo, and iodo.

Heteroaryl: an aromatic group of from 1 to 10 carbon atoms and 1 to 4heteroatoms selected from the group consisting of oxygen, nitrogen, andsulfur within the ring. Such heteroaryl groups can have a single ring(e.g., pyridinyl or furyl) or multiple condensed rings (e.g.,indolizinyl or benzothienyl), wherein the condensed rings may or may notbe aromatic and/or contain a heteroatom, provided that the point ofattachment is through an atom of the aromatic heteroaryl group. In oneembodiment, the nitrogen and/or sulfur ring atom(s) of the heteroarylgroup are optionally oxidized to provide for the N-oxide (N→O),sulfinyl, or sulfonyl moieties.

Heteroaryloxy: —O-heteroaryl.

Heteroarylthio: —S-heteroaryl. This term also encompasses oxidized formsof sulfur, such as —S(O)— heteroaryl, or —S(O)₂-heteoaryl.

Heterocyclyl: a saturated, unsaturated group, or combinations thereof,having a single ring or multiple condensed rings, including fusedbridged and spiro ring systems, and having from 3 to 15 ring atoms,including 1 to 4 heteroatoms, selected from nitrogen, sulfur, or oxygen.These groups may be substituted with one or more of the substituentsdisclosed herein for substituted aryl and/or substituted alkyl. Thesegroups encompass, for example, a saturated heterocyclyl fused with oneor more aromatic hydrocarbons or heteroaryl groups.

Heterocyclyloxy: —O-heterocycyl.

Heterocyclylthio: —S-heterocycyl. This term also encompasses oxidizedforms of sulfur, such as —S(O)-heterocyclyl, or —S(O)₂-heterocyclyl.

Hydroxyl or Hydroxy: —OH.

Imino: —N═R^(c) wherein R^(c) may be selected from hydrogen,aminocarbonylalkyloxy, substituted aminocarbonylalkyloxy,aminocarbonylalkylamino, and substituted aminocarbonylalkylamino.

Increase: To enhance the quality, amount, or strength of something. Inone example, an agent increases the activity or expression of α7β1, forexample relative to an absence of the agent. In a particular example, anagent increases the activity or expression of α7β1 by at least 10%, atleast 20%, at least 50%, or even at least 90%, including between 10% to95%, 20% to 80%, 30% to 70%, 40% to 50%, such as 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100%. Such increases can bemeasured using the methods disclosed herein.

In a particular example, a therapy increases (also known asup-regulates) the expression of α7β1, such as an increase of at least10%, at least 20%, at least 50%, or even at least 90% in α7β1expression, thereby treating/alleviating one or more signs or symptomsassociated with muscular dystrophy. In some examples, an increase inexpression refers to an increase in a α7β1 gene product. An α7β1 geneproduct can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) orprotein.

Gene upregulation includes any detectable increase in the production ofa α7β1 gene product. In certain examples, production of a α7β1 geneproduct increases by at least 2-fold, for example at least 3-fold or atleast 4-fold, as compared to a control (such an amount of geneexpression in a normal cell). In one example, a control is a relativeamount of α7 gene expression or protein expression in a biologicalsample taken from a subject who does not have muscular dystrophy, suchas DMD, FCMD or MDC1A. Such increases can be measured using the methodsdisclosed herein. For example, “detecting or measuring expression ofα7β1” includes quantifying the amount of the gene, gene product ormodulator thereof present in a sample. Quantification can be eithernumerical or relative. Detecting expression of the gene, gene product ormodulators thereof can be achieved using any method known in the art ordescribed herein, such as by measuring nucleic acids by PCR (such asRT-PCR) and proteins by ELISA. In primary embodiments, the changedetected is an increase or decrease in expression as compared to acontrol, such as a reference value or a healthy control subject. In someexamples, the detected increase or decrease is an increase or decreaseof at least two-fold compared with the control or standard. Controls orstandards for comparison to a sample, for the determination ofdifferential expression, include samples believed to be normal (in thatthey are not altered for the desired characteristic, for example asample from a subject who does not have muscular dystrophy, such as DMD,FCMD or MDC1A) as well as laboratory values (e.g., range of values),even though possibly arbitrarily set, keeping in mind that such valuescan vary from laboratory to laboratory.

Laboratory standards and values can be set based on a known ordetermined population value and can be supplied in the format of a graphor table that permits comparison of measured, experimentally determinedvalues.

In other embodiments of the methods, the increase or decrease is of adiagnostically significant amount, which refers to a change of asufficient magnitude to provide a statistical probability of thediagnosis.

The level of expression in either a qualitative or quantitative mannercan detect nucleic acid or protein. Exemplary methods include microarrayanalysis, RT-PCR, Northern blot, Western blot, and mass spectrometry.

Inhibiting a disease or condition: A phrase referring to reducing thedevelopment of a disease or condition, for example, in a subject who isat risk for a disease or who has a particular disease. Particularmethods of the present disclosure provide methods for inhibitingmuscular dystrophy.

Integrin: A cell surface transmembrane glycoprotein receptor. Integrinsare involved in many biological processes such as wound healing, bloodclot formation, gene regulation, and immune responses.

Integrins can regulate tissue specific cell adhesion molecules.Integrins are heterodimeric non-covalently associated glycoproteinscomposed of two subunits. The subunits, which are designated α and beta,have approximate molecular weights of 150-180 kilodaltons and 90-110kilodaltons, respectively.

The α7β1 integrin is a major laminin receptor expressed in skeletalmuscle. The α7β1 integrin plays a role in the development ofneuromuscular and myotendinous junctions. In the adult, the α7β1integrin is concentrated at junctional sites and found inextrajunctional regions where it mediates the adhesion of the musclefibers to the extracellular matrix. Mice that lack the α7 chain developmuscular dystrophy that affects the myotendinous junctions. The absenceof α7 integrin results in defective matrix deposition at themyotendinous junction. Loss of the α7 integrin in γ-sarcoglycan miceresults in severe muscle pathology. Absence of the α7 integrin in mdxmice also results in severe muscular dystrophy, confirming that the α7β1integrin serves as a major genetic modifier for Duchenne and othermuscular dystrophies.

Mutations in the α7 gene are responsible for muscular dystrophy inhumans. A screen of 117 muscle biopsies from patients with undefinedmuscle disease revealed 3 which lacked the α7 integrin chain and hadreduced levels of (31D integrin chain. These patients exhibited delayeddevelopmental milestones and impaired mobility consistent with the rolefor the α7β1 integrin in neuromuscular and myotendinous junctiondevelopment and function.

Several lines of evidence suggest the α7 integrin may be important formuscle regeneration. For example, during embryonic development, the α7β1integrin regulates myoblast migration to regions of myofiber formation.It has been found that MyoD (myogenic determination protein)transactivates α7 integrin gene expression in vitro, which wouldincrease α7 integrin levels in activated satellite cells. Human, mouseand rat myoblast cell lines derived from satellite cells express highlevels of α7 integrin. Elevated α7 integrin mRNA and protein aredetected in the skeletal muscle of 5 week old mdx mice, which correlateswith the period of maximum muscle degeneration and regeneration. Inaddition, the α7β1 integrin associates with muscle specific β1-integrinbinding protein (MIBP), which regulates laminin deposition in C2C12myoblasts. Laminin provides an environment that supports myoblastmigration and proliferation. Finally, enhanced expression of the α7integrin in dystrophic skeletal muscle results in increased numbers ofsatellite cells.

The sequences for α7β1 integrin subunits are publicly available onGenBank, see, for example Gene Accession No. NM_001144116 (human) andNM_008398.2 (mouse) for α7 integrin, and Gene Accession No. NM_002211for β1 integrin (also known as CD29), each of which is hereinincorporated by reference as available on Sep. 8, 2011. Exemplary α7β1integrin modulatory agents are disclosed herein, such as a compoundencompassed by any one of Formulas 1-16, or provided by any one ofTables 1-16 and 18.

A α7β1 integrin-associated condition is a condition associated withaltered α7β1 integrin expression or activity, including musculardystrophy, such as DMD, FCMD, LGMD, FHMD, Beckers muscular dystrophyand/or MDC1A.

Laminin: Any of the family of glycoproteins that are typically involvedin the formation and maintenance of extracellular matrices. Laminin is aheterotrimers formed from an α chain, a chain, and a γ chain. Thevarious chains of a particular laminin can affect the properties of themolecule. In some aspects of the present disclosure, fragments,derivatives, or analogs of various laminins can be used, such aslaminins having at least a portion at least substantially homologous tothe laminin α1 chain. A “fragment of laminin,” as used herein, refers toa portion of a substance, such as laminin. A fragment may be, in someexamples, a particular domain or chain of a protein. For example,particular embodiments of the present disclosure involve administering afragment of laminin-1 corresponding to at least a portion of (or all of)the laminin α1 chain. Fragments may be synthetic or may be derived fromlarger parent substances.

In some aspects, laminins may be administered as a mixture of laminins,including fragments, analogs, and derivatives thereof. Suitable methodsfor preparing analogs of laminin domains are disclosed in U.S. Pat. No.6,933,280, incorporated by reference herein to the extent notinconsistent with this disclosure.

The laminin materials or compositions of the present disclosure may bedelivered as discrete molecules or may be complexed with, or conjugatedto, another substance. For example, the laminin may be combined with acarrier, such as to aid in delivery of the laminin to a site of interestor to increase physiological uptake or incorporation of the laminin.

In specific examples, the laminin administered includes or consists oflaminin-1 (LAM-111), which includes the chains α1β1γ1. In furtherexamples, the laminin administered includes or consists of laminin-2,which includes the chains α2β1γ1. In yet further examples, the lamininadministered includes or consists of laminin-4, which includes thechains α2β2γ1.

Laminins may be obtained from any suitable source. For example,laminin-1 may be obtained from placental tissue or fromEngelbreth-Holm-Swarm murine sarcoma. Suitable methods of isolatingvarious laminins are disclosed in U.S. Pat. No. 5,444,158, incorporatedby reference herein to the extent not inconsistent with the presentdisclosure.

Muscle: Any myoblast, myocyte, myofiber, myotube or other structurecomposed of muscle cells. Muscles or myocytes can be skeletal, smooth,or cardiac. Muscle may also refer to, in particular implementations ofthe present disclosure, cells or other materials capable of formingmyocytes, such as stem cells and satellite cells.

Muscular dystrophy: A term used to refer to a group of genetic disordersthat lead to progressive muscle weakness. Muscular dystrophy can resultin skeletal muscle weakness and defects in skeletal muscle proteins,leading to a variety of impaired physiological functions. Nosatisfactory treatment of muscular dystrophy exists. Existing treatmentstypically focus on ameliorating the effects of the disease and improvingthe patient's quality of life, such as through physical therapy orthrough the provision of orthopedic devices.

Mutated genes associated with muscular dystrophy are responsible forencoding a number of proteins associated with the costameric proteinnetwork. Such proteins include laminin-2, collagen, dystroglycan,integrins, caveolin-3, ankyrin, dystrophin, α-dystrobrevin, vinculin,plectin, BPAG1b, muscle LIM protein, desmin, actinin-associated LIMprotein, α-actin, titin, telethonin, cypher, myotilin, and thesarcoglycan/sarcospan complex.

The most common form of muscular dystrophy is DMD, affecting 1 in 3,500live male births. DMD is an X-linked recessive disorder characterized bya mutation in the gene that codes for dystrophin. Dystrophin is acytoskeletal protein about 430 kDa in size. This protein works toconnect the cell's cytoskeleton and extracellular matrix. The loss ofdystrophin in DMD patients leads to a loss of muscle fiber attachment atthe extracellular matrix during contraction, which ultimately leads toprogressive fiber damage, membrane leakage and a loss of musclefunction. Most patients die before they reach the age of 30 due torespiratory or cardiac failure.

Beckers muscular dystrophy (also known as Benign pseudohypertrophicmuscular dystrophy) is related to DMD in that both result from amutation in the dystrophin gene, but in DMD no functional dystrophin isproduced making DMD much more severe than BMD. BMD is an X-linkedrecessive inherited disorder characterized by slowly progressive muscleweakness of the legs and pelvis. BMD is a type of dystrophinopathy,which includes a spectrum of muscle diseases in which there isinsufficient dystrophin produced in the muscle cells, results ininstability in the structure of muscle cell membrane. This is caused bymutations in the dystrophin gene, which encodes the protein dystrophin.The pattern of symptom development of BMD is similar to DMD, but with alater, and much slower rate of progression.

Congenital muscular dystrophies are caused by gene mutations. FCMD andMDC1A are examples of congenital muscular dystrophies. MDC1A is acongenital muscular dystrophy due to a genetic mutation in the LAMA2gene which results in lack of or complete loss of laminin-α2 protein.This loss of laminin-α2 leads to an absence of laminins-211/221.Laminins-211/221 are major components of the extracellular matrix andplay a key role in muscle cell development. During muscle celldifferentiation laminin binds to the α7β1 integrin. Without laminin-α2,muscle fibers are unable to adhere to the basement membrane and myotubesundergo apotosis. Muscle regeneration also fails, leading to a loss ofmuscle repair and an increase in muscle fibrosis and inflammation. Thischronic tissue injury is a major cause of morbidity and mortality inMDC1A.

Congenital Muscular Dystrophies (CMD) and Limb-Girdle muscular dystrophy(LGMD) are common forms of highly heterogeneous muscular dystrophieswhich can be distinguished by their age at onset. In CMD, onset ofsymptoms is at birth or within the first 6 months of life; in LGMD onsetof symptoms is in late childhood, adolescence or even adult life.Inheritance in LGMD can be autosomal dominant (LGMD type 1) or autosomalrecessive (LGMD type 2), CMD is recessively inherited. CMD and LGMD canoverlap both clinically and genetically

MDC1A is a progressive muscle wasting disease that results in childrenbeing confined to a wheelchair, requiring ventilator assistance tobreathe and premature death. Symptoms are detected at birth with poormuscle tone and “floppy” baby syndrome. DMD, BMD and LGMD areprogressive muscle degenerative diseases usually diagnosed at 3-5 yearsof age when children show developmental delay including ability to walkand climb stairs. The disease is progressive and children are usuallyconfined to a wheelchair in their teens and require ventilatorassistance.

Fukuyama congenital muscular dystrophy (FCMD) is an inherited conditionthat predominantly affects the muscles, brain, and eyes. Congenitalmuscular dystrophies are a group of genetic conditions that cause muscleweakness and wasting (atrophy) beginning very early in life. Fukuyamacongenital muscular dystrophy affects the skeletal muscles, which aremuscles the body uses for movement. The first signs of the disorderappear in early infancy and include a weak cry, poor feeding, and weakmuscle tone (hypotonia). Weakness of the facial muscles often leads to adistinctive facial appearance including droopy eyelids (ptosis) and anopen mouth. In childhood, muscle weakness and joint deformities(contractures) restrict movement and interfere with the development ofmotor skills such as sitting, standing, and walking. Fukuyama congenitalmuscular dystrophy also impairs brain development. People with thiscondition have a brain abnormality called cobblestone lissencephaly, inwhich the surface of the brain develops a bumpy, irregular appearance(like that of cobblestones). These changes in the structure of the brainlead to significantly delayed development of speech and motor skills andmoderate to severe intellectual disability. Social skills are lessseverely impaired. Most children with Fukuyama congenital musculardystrophy are never able to stand or walk, although some can sit withoutsupport and slide across the floor in a seated position. More than halfof all affected children also experience seizures. Other signs andsymptoms of Fukuyama congenital muscular dystrophy include impairedvision, other eye abnormalities, and slowly progressive heart problemsafter age 10. As the disease progresses, affected people may developswallowing difficulties that can lead to a bacterial lung infectioncalled aspiration pneumonia. Because of the serious medical problemsassociated with Fukuyama congenital muscular dystrophy, most people withthe disorder live only into late childhood or adolescence.

Fukuyama congenital muscular dystrophy is seen almost exclusively inJapan, where it is the second most common form of childhood musculardystrophy (after Duchenne muscular dystrophy). Fukuyama congenitalmuscular dystrophy has an estimated incidence of 2 to 4 per 100,000Japanese infants.

Fukuyama congenital muscular dystrophy is caused by mutations in theFKTN gene which encodes fukutin. The most common mutation in the FKTNgene reduces the amount of fukutin produced within cells. A shortage offukutin likely prevents the normal modification of α-dystroglycan, whichdisrupts that protein's normal function. Without functionalα-dystroglycan to stabilize muscle cells, muscle fibers become damagedas they repeatedly contract and relax with use. The damaged fibersweaken and die over time, leading to progressive weakness and atrophy ofthe skeletal muscles.

Defective α-dystroglycan also affects the migration of neurons duringthe early development of the brain. Instead of stopping when they reachtheir intended destinations, some neurons migrate past the surface ofthe brain into the fluid-filled space that surrounds it. BecauseFukuyama congenital muscular dystrophy involves a malfunction ofα-dystroglycan, this condition is described as a dystroglycanopathy.

Facioscapulohumeral muscular dystrophy (FHMD) is a form of musculardystrophy associated with progressive muscle weakness and loss of muscletissue. Unlike DMD and BMD which mainly affect the lower body, FSHDaffects the upper body mainly the face, shoulder and upper arm muscles.However, it can affect muscles around the pelvis, hips, and lower leg.Symptoms for FSHD often do not appear until age 10-26, but it is notuncommon for symptoms to appear much later. In some cases, symptomsnever develop. Symptoms are usually mild and very slowly become worse.Facial muscle weakness is common, and may include eyelid drooping,inability to whistle, decreased facial expression, depressed or angryfacial expression, difficulty pronouncing words, shoulder muscleweakness (leading to deformities such as pronounced shoulder blades(scapular winging) and sloping shoulders), weakness of the lower,hearing loss and possible heart conditions.

Oxo: (═O).

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or more agents,such as one or more α7β1 modulatory agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationscan include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In addition to biologically-neutral carriers, pharmaceuticalagents to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate, sodium lactate, potassium chloride,calcium chloride, and triethanolamine oleate.

Sample (or biological sample): A biological specimen containing genomicDNA, RNA (including mRNA), protein, or combinations thereof, obtainedfrom a subject. Examples include, but are not limited to, peripheralblood, urine, saliva, tissue biopsy, surgical specimen, and autopsymaterial. In one example, a sample includes muscle biopsy, such as froma subject with DMD, FCMD, or MDC1A.

Signs or symptoms: Any subjective evidence of disease or of a subject'scondition, e.g., such evidence as perceived by the subject; a noticeablechange in a subject's condition indicative of some bodily or mentalstate. A “sign” is any abnormality indicative of disease, discoverableon examination or assessment of a subject. A sign is generally anobjective indication of disease. Signs include, but are not limited toany measurable parameters such as tests for detecting musculardystrophy, including measuring creatine kinase levels, electromyography(to determine if weakness is caused by destruction of muscle tissuerather than by damage to nerves) orimmunohistochemistry/immunoblotting/immunoassay (e.g., ELISA) to measuremuscular dystrophy-associated molecules, such as α7β1 integrin. In oneexample, reducing or inhibiting one or more symptoms or signs associatedwith muscular dystrophy, includes increasing the activity or expressionof α7β1 integrin by a desired amount, for example by at least 20%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or even at least 100%, as compared to theactivity and/or expression in the absence of the treatment. Symptoms ofmuscular dystrophy include, but are not limited to, muscle weakness andloss, difficulty running, difficulty hopping, difficulty jumping,difficulty walking, difficulty breathing, fatigue, skeletal deformities,muscle deformities (contractions of heels; pseudohypertrophy of calfmuscles), heart disease (such as dilated cardiomyopathy), elevatedcreatine phosphokinase (CK) levels in blood or combinations thereof.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals.

Substituted Alkyl: an alkyl (or alkenyl, or alkynyl) group having from 1to 5 hydrogen atoms replaced with substituents selected alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, acylalkyloxy, amino,substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy,aminodicarbonylamino, aminocarbonylalkyl, aminosulfonyl, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, aminodiacylamino,cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substitutedcycloalkylthio, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclyl, substituted heterocyclyl, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, imino, oxo, sulfonylamino, nitro, SO₃H, sulfonyl,thiol, imino, substituted imino, alkylthio, and substituted alkylthio.The alkyl may be substituted with 1 to 2, 1 to 3, or 1 to 4 of thesegroups, which are defined herein.

Substituted Alkoxy: —O-(substituted alkyl).

Substituted Alkylthio: —S-(substituted alkyl). This term alsoencompasses oxidized forms of sulfur, such as —S(O)-substituted alkyl,or —S(O)₂-substituted alkyl.

Substituted Amino: —N(R^(b))₂, wherein each R^(b) independently isselected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) mayoptionally be joined together with the nitrogen bound thereto to form aheterocyclyl or substituted heterocyclyl group, provided that both R^(b)are not both hydrogen.

Substituted Aryl: aryl groups having 1 to 5 hydrogens replaced withsubstituents independently selected from alkoxy, substituted alkoxy,acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aminosulfonyl, aryl, substitutedaryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio,carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy,cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, halo,hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substitutedheteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy,heterocyclylthio, substituted heterocyclylthio, nitro, SO₃H, sulfonyl,thiol, alkylthio, and substituted alkylthio. The aryl group may besubstituted with 1 to 2, 1 to 3, or 1 to 4 of these groups, which aredefined herein.

Substituted Aryloxy: —O-(substituted aryl).

Substituted Arylthio: —S-(substituted aryl), wherein substituted aryl isas defined herein. This term also encompasses oxidized forms of sulfur,such as —S(O)-substituted aryl, or —S(O)₂-substituted aryl.

Substituted Cycloalkyl: cycloalkyl, cycloalkenyl, or cycloalkynyl grouphaving from 1 to 5 substituents selected from the group consisting ofoxo, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy,aminosulfonyl, aryl, substituted aryl, aryloxy, substituted aryloxy,arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxylester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substitutedcycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,substituted cycloalkylthio, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclyl, substituted heterocyclyl,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, sulfonyl, thiol, alkylthio,and substituted alkylthio. The aryl group may be substituted with 1 to2, 1 to 3, or 1 to 4 of these groups, which are defined herein. In someembodiments, the cycloalkyl group may have multiple condensed rings(e.g. tetrahydronaphthyl or tetrahydroanthacenyl), provided that thepoint of attachment is through an atom of the nonaromatic ring.

Substituted (Cycloalkyl)oxy: —O-(substituted cycloalkyl).

Substituted (Cycloalkyl)thio: refers to —S-(substituted cycloalkyl).This term also encompasses oxidized forms of sulfur, such as—S(O)-substituted cycloalkyl, or —S(O)₂-substituted cycloalkyl.

Substituted Heteroaryl: heteroaryl groups that are substituted with from1 to 5 substituents selected from the group consisting of the same groupof substituents defined for substituted aryl.

Substituted Heteroaryloxy: —O-(substituted heteroaryl).

Substituted Heteroarylthio: —S-(substituted heteroaryl). This term alsoencompasses oxidized forms of sulfur, such as —S(O)-substitutedheteroaryl, or —S(O)₂-substituted heteoaryl.

Substituted Heterocycyloxy: —O-(substituted heterocyclyl) wherein theheterocyclyl group is substituted with one or more of the substituentsrecited for substituted alkyl.

Substituted Heterocycylthio: —S-(substituted heterocycyl). This termalso encompasses oxidized forms of sulfur, such as —S(O)-substitutedheterocyclyl, or —S(O)₂-substituted heterocyclyl.

Sulfonyl: —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-cycloalkyl,—SO₂-substituted cycloalkyl, —SO₂-aryl, —SO₂-substituted aryl,—SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclyl, and—SO₂-substituted heterocyclyl.

Sulfonylamino: —NR^(a)SO₂alkyl, —NR^(a)SO₂ substituted alkyl,—NR^(a)SO₂cycloalkyl, —NR^(a)SO₂ substituted cycloalkyl, —NR^(a)SO₂aryl,—NR^(a)SO₂ substituted aryl, —NR^(a)SO₂heteroaryl, —NR^(a)SO₂substituted heteroaryl, —NR^(a)SO₂heterocyclyl, —NR^(a)SO₂ substitutedheterocyclyl, wherein each R^(a) independently is as defined herein.

Thiol: —SH.

Thiocarbonyl: (═S)

Tissue: An aggregate of cells, usually of a particular kind, togetherwith their intercellular substance that form one of the structuralmaterials of an animal and that in animals include connective tissue,epithelium, muscle tissue, and nerve tissue.

Treating a disease: A therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition related to a musculardystrophy, such as a sign or symptom of muscular dystrophy. Treatmentcan induce remission or cure of a condition or slow progression, forexample, in some instances can include inhibiting the full developmentof a disease, for example preventing development of a musculardystrophy. Prevention of a disease does not require a total absence ofdisease. For example, a decrease of at least 50% can be sufficient.

Treating a disease can be a reduction in severity of some or allclinical symptoms of the disease or condition, a reduction in the numberof relapses of the disease or condition, an improvement in the overallhealth or well-being of the subject, by other parameters well known inthe art that are specific to the particular disease or condition, andcombinations of such factors.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. In one example, includesadministering a disclosed agent to a subject sufficient to allow thedesired activity. In particular examples, the desired activity isincreasing the expression or activity of α7β1.

III. Compounds for Treating Muscular Dystrophy

Disclosed herein are compounds that may be used as α1β7 integrinmodulatory agents in methods disclosed herein. In particular disclosedembodiments, the compound is effective in treating muscular dystrophy.The compound is a small-molecule therapeutic. In particular disclosedembodiments, the small-molecule therapeutic is a cyclic compoundcomprising a heteroatom-containing skeleton. In other disclosedembodiments, the small-molecule therapeutic is a cyclic compoundcomprising an all-carbon skeleton. In certain disclosed embodiments, thecyclic compound comprising a heteroatom-containing skeleton has aformula illustrated below:

wherein each R¹ independently is selected from C₁₋₁₀alkyl, substitutedC₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino,acyloxy, acylC₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl,aminocarbonylC₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino,aminocarbonyloxy, aminosulfonyl, C₆₋₁₅ aryl, substituted C₆₋₁₅ aryl,C₆₋₁₅ aryloxy, substituted C₆₋₁₅ aryloxy, C₆₋₁₅ arylthio, substitutedC₆₋₁₅ arylthio, carboxyl, carboxyester, (carboxyester)amino,(carboxyester)oxy, cyano, C₃₋₈ cycloalkyl, substituted C₃₋₈ cycloalkyl,(C₃₋₈ cycloalkyl)oxy, substituted (C₃₋₈ cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈ cycloalkyl)thio, halo, hydroxyl,C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy,substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substitutedC₁₋₁₀heteroarylthio, C₂₋₁₀ heterocyclyl, C₂₋₁₀ substituted heterocyclyl,C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, and substitutedC₁₋₁₀alkythio, thiocarbonyl; or

two R¹ substituents, together with the atom to which each is bound, mayform ring selected from a C₆₋₁₅aryl, substituted C₆₋₁₅ aryl, C₃₋₈cycloalkyl, substituted C₃₋₈ cycloalkyl, C₁₋₁₀heteroaryl, substitutedC₁₋₁₀heteroaryl, C₂₋₁₀ substituted heterocyclyl, andC₂₋₁₀heterocyclyloxy, substituted;

each of A, B, C, D, and E independently may be selected from carbon,nitrogen, oxygen, and sulfur; and

n may be zero, 1, 2, 3, 4, or 5.

In other embodiments, the cyclic compound comprising aheteroatom-containing moiety has a formula illustrated below:

wherein each R² independently is selected from C₁₋₁₀alkyl, substitutedC₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino,acyloxy, acylC₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl,aminocarbonylC₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino,aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅ aryl,C₆₋₁₅ aryloxy, substituted C₆₋₁₅ aryloxy, C₆₋₁₅ arylthio, substitutedC₆₋₁₅ arylthio, carboxyl, carboxyester, (carboxyester)amino,(carboxyester)oxy, cyano, C₃₋₈ cycloalkyl, substituted C₃₋₈ cycloalkyl,(C₃₋₈ cycloalkyl)oxy, substituted (C₃₋₈ cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈ cycloalkyl)thio, halo, hydroxyl,C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy,substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substitutedC₁₋₁₀heteroarylthio, C₂₋₁₀ heterocyclyl, C₂₋₁₀ substituted heterocyclyl,C₂₋₁₀ heterocyclyloxy, substituted C₂₋₁₀ heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀ heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, and substitutedC₁₋₁₀alkythio, thiocarbonyl; or

two R² substituents, together with the atom to which each is bound, mayform ring selected from a C₆₋₁₅aryl, substituted C₆₋₁₅ aryl, C₃₋₈cycloalkyl, substituted C₃₋₈ cycloalkyl, C₁₋₁₀heteroaryl, substitutedC₁₋₁₀heteroaryl, C₂₋₁₀ substituted heterocyclyl, andC₂₋₁₀heterocyclyloxy, substituted;

each of A, B, C, D, E, and F independently may be selected from carbon,nitrogen, oxygen, and sulfur; and

n may be zero, 1, 2, 3, 4, or 5.

In particular disclosed embodiments, the cyclic compound comprising anall-carbon skeleton may have a general formula provided below:

wherein R³ and R⁴ independently may be selected from hydroxyl, hydrogen,C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, carboxyl, acyl, aminoacyl,acylamino, amino, substituted amino, C₆₋₁₅ aryl, substituted C₆₋₁₅ aryl,and C₁₋₁₀alkoxy; R⁵ is selected from amino, substituted amino, oxo,hydroxyl, C₁₋₁₀alkoxy, and imino; and n may be zero, 1, 2, 3, 4, or 5. Aperson of ordinary skill in the art will recognize that the dashed linesindicate optional bonds which may be present in certain compounds andnot present in others.

In particular disclosed embodiments, rings A and B are connected via theoptional bonds to form a steroid-based skeleton. In embodiments whereinrings A and B are connected, R⁵ may be bound to ring A via a double bondor a single bond, a feature that is indicated with the optional dashedbond in Formula 13. For example, if R⁵ is amino, hydroxyl, substitutedamino, or C₁₋₁₀alkoxy, then R⁵ is attached to ring A via a single bond,whereas if R⁵ is oxo or imino, then R5 is attached to ring A via adouble bond.

In particular disclosed embodiments, C₆₋₁₅ aryl may be selected fromphenyl, biphenyl, naphthalene, anthracene, and the like; substitutedC₆₋₁₅ aryl may be selected from phenyl, biphenyl, naphthalene, andanthracene substituted with one or more substituents as defined herein;C₁₋₁₀alkyl may be selected from C₁₋₁₀alkane, C₂₋₁₀alkene, andC₂₋₁₀alkyne; more typically from methyl, ethyl, propyl, butyl, pentyl,hexyl, and the like; ethylene, propylene, butylene, and the like; andethyne, propyne, butyne, and the like; substituted C₁₋₁₀alkyl may beselected from C₁₋₁₀alkane, C₂₋₁₀alkene, and C₂₋₁₀alkyne substituted withone or of the substituents as provided herein.

Exemplary embodiments concerning heterocyclyl and heteroarylsubstitutents include, but are not limited to, epoxy, pyrrolyl,imidazole, pyrazole, pyridinyl, pyrazine, pyrimidine, oxanyl, thianyl,dioxanyl, dithianyl, coumarin, pyridazine, indolizine, isoindole,indolyl, indolinyl (or dihydroindole), indazole, purine, isoquinoline,quinoline, benzo[d]pyridazine, naphthyridine, quinoxaline, quinazoline,benzopyridazine, pteridine, carbazole, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, indoline, phthalimide,1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene,thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl,thiomorpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, oxazolidinyl,oxazolyl, thiophenyl, isooxazolidinyl, and tetrahydrofuranyl.

Exemplary substituents wherein at least two R¹ groups have been joinedtogether include the following:

Particular disclosed embodiments concern cyclic compounds comprising afive-membered heteroatom-containing skeleton having a formula selectedfrom those provided below.

With reference to Formulas 4-6, R¹ and n are as recited herein, and eachX independently may be selected from carbon, oxygen, nitrogen, andsulfur.

In yet other embodiments, the cyclic compound comprising a five-memberedheteroatom-containing skeleton may have any one of the followingformulas

wherein R′ is as recited herein.

Exemplary compounds are provided below.

TABLE 1 Exemplary Compounds

Particular embodiments concern cyclic compounds comprising asix-membered heteroatom-containing skeleton having any one of theformulas provided below:

wherein R² and n are as recited herein, Z may be selected from carbonand nitrogen, Y may be selected from nitrogen and oxygen, and each Xindependently may be selected from nitrogen and carbon. A person ofordinary skill in the art will recognize that the dashed lines indicatevariable bonds which may or may not be present, depending on the valencyof the atom to which each variable bond is attached. For example, if thevariable bond indicated in Formula 11 is present, X typically is carbon,as a carbon atom can accommodate four bonds. X may be nitrogen in such acompound; however, a person of ordinary skill in the art would recognizethat the nitrogen atom would be positively charged due to the fact thatits lone pairs are used to accommodate a fourth bond.

Exemplary compounds are provided below solely as illustrative examples.

TABLE 2 Exemplary Compounds

Particular embodiments concern compounds comprising an all-carbon,steroidal skeleton having a formula as illustrated below.

An exemplary compound is provided below.

In other disclosed embodiments of the compound comprising an all-carbonskeleton, ring A is not connected with ring B and exists as an arylcompound having a formula illustrated below.

Exemplary compounds are illustrated below.

Other compounds suitable for use in the methods disclosed herein areprovided below in Tables 3, 4, 5, and 6. Any of the compounds providedin Tables 3-6, and any other compounds disclosed herein, can be madeusing synthetic methods well known in the art of chemical synthesis.

TABLE 3

R¹ R^(1′) R² R³ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo Hor —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ HaloH or —(CH₂)₀₋₅CH₃ H NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃C(O)O(CH₂)₀₋₅CH₃ NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)OHNHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂OH H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OHH or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or—(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or—(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH Hor —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH Hor —(CH₂)₀₋₅CH₃ H NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃C(O)O(CH₂)₀₋₅CH₃ NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)OHNHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³,or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or—(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂—O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂—O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ Hor —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ Hor —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenylNHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or—(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or—(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or—(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ Hor —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or—(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂—(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenylNHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂—(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPhNHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or—(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, orN[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, orN[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ Hor —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or—(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or—(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ Hor —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ Hor —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃

TABLE 4

R¹ R² C(O)NH₂ —Ph-Halo C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph-Halo C(O)NHPh —Ph-HaloC(O)NH-2,4-fluorophenyl —Ph-Halo C(O)NH-4-fluorophenyl —Ph-HaloC(O)NH-3-fluorophenyl —Ph-Halo C(O)NH-4-morpholinylphenyl —Ph-HaloC(O)NH-4-piperazinyllphenyl —Ph-Halo C(O)NH-3-thiomethylphenyl —Ph-HaloH —Ph-Halo C(O)O(CH₂)₀₋₅CH₃ —Ph-Halo C(O)OH —Ph-Halo Ph—OH —Ph-HaloPh—NH₂ —Ph-Halo Ph-Halo —Ph-Halo Ph—O(CH₂)₀₋₅CH₃ —Ph-HaloPh—N[(CH₂)₀₋₅CH₃]₂ —Ph-Halo C(O)NH₂ —Ph—OH C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—OHC(O)NHPh —Ph—OH C(O)NH-2,4-fluorophenyl —Ph—OH C(O)NH-4-fluorophenyl—Ph—OH C(O)NH-3-fluorophenyl —Ph—OH C(O)NH-4-morpholinylphenyl —Ph—OHC(O)NH-4-piperazinyllphenyl —Ph—OH C(O)NH-3-thiomethylphenyl —Ph—OH H—Ph—OH C(O)O(CH₂)₀₋₅CH₃ —Ph—OH C(O)OH —Ph—OH Ph—OH —Ph—OH Ph—NH₂ —Ph—OHPh-Halo —Ph—OH Ph—O(CH₂)₀₋₅CH₃ —Ph—OH Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—OH C(O)NH₂—Ph—O(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—O(CH₂)₀₋₅CH₃ C(O)NHPh—Ph—O(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl —Ph—O(CH₂)₀₋₅CH₃C(O)NH-4-fluorophenyl —Ph—O(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl—Ph—O(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl —Ph—O(CH₂)₀₋₅CH₃C(O)NH-4-piperazinyllphenyl —Ph—O(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl—Ph—O(CH₂)₀₋₅CH₃ H —Ph—O(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ —Ph—O(CH₂)₀₋₅CH₃C(O)OH —Ph—O(CH₂)₀₋₅CH₃ Ph—OH —Ph—O(CH₂)₀₋₅CH₃ Ph—NH₂ —Ph—O(CH₂)₀₋₅CH₃Ph-Halo —Ph—O(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ —Ph—O(CH₂)₀₋₅CH₃Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—O(CH₂)₀₋₅CH₃ C(O)NH₂ —Ph—NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂—Ph—NH₂ C(O)NHPh —Ph—NH₂ C(O)NH-2,4-fluorophenyl —Ph—NH₂C(O)NH-4-fluorophenyl —Ph—NH₂ C(O)NH-3-fluorophenyl —Ph—NH₂C(O)NH-4-morpholinylphenyl —Ph—NH₂ C(O)NH-4-piperazinyllphenyl —Ph—NH₂C(O)NH-3-thiomethylphenyl —Ph—NH₂ H —Ph—NH₂ C(O)O(CH₂)₀₋₅CH₃ —Ph—NH₂C(O)OH —Ph—NH₂ Ph—OH —Ph—NH₂ Ph—NH₂ —Ph—NH₂ Ph-Halo —Ph—NH₂Ph—O(CH₂)₀₋₅CH₃ —Ph—NH₂ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—NH₂ C(O)NH₂—Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NHPh—Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH-2,4-fluorophenyl —Ph—N[(CH₂)₀₋₅CH₃]₂C(O)NH-4-fluorophenyl —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH-3-fluorophenyl—Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-morpholinylphenyl —Ph—N[(CH₂)₀₋₅CH₃]₂C(O)NH-4-piperazinyllphenyl —Ph—N[(CH₂)₀₋₅CH₃]₂C(O)NH-3-thiomethylphenyl —Ph—N[(CH₂)₀₋₅CH₃]₂ H —Ph—N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)OH —Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—OH—Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—NH₂ —Ph—N[(CH₂)₀₋₅CH₃]₂ Ph-Halo—Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—O(CH₂)₀₋₅CH₃ —Ph—N[(CH₂)₀₋₅CH₃]₂Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ —Ph—(CH₂)₀₋₅CH₃C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—(CH₂)₀₋₅CH₃ C(O)NHPh —Ph—(CH₂)₀₋₅CH₃C(O)NH-2,4-fluorophenyl —Ph—(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl—Ph—(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl —Ph—(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl —Ph—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl—Ph—(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl —Ph—(CH₂)₀₋₅CH₃ H—Ph—(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ —Ph—(CH₂)₀₋₅CH₃ C(O)OH —Ph—(CH₂)₀₋₅CH₃Ph—OH —Ph—(CH₂)₀₋₅CH₃ Ph—NH₂ —Ph—(CH₂)₀₋₅CH₃ Ph-Halo —Ph—(CH₂)₀₋₅CH₃Ph—O(CH₂)₀₋₅CH₃ —Ph—(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—(CH₂)₀₋₅CH₃C(O)NH₂ —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—S[(CH₂)₀₋₅CH₃]₂C(O)NHPh —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH-2,4-fluorophenyl —Ph—S[(CH₂)₀₋₅CH₃]₂C(O)NH-4-fluorophenyl —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH-3-fluorophenyl—Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-morpholinylphenyl —Ph—S[(CH₂)₀₋₅CH₃]₂C(O)NH-4-piperazinyllphenyl —Ph—S[(CH₂)₀₋₅CH₃]₂C(O)NH-3-thiomethylphenyl —Ph—S[(CH₂)₀₋₅CH₃]₂ H —Ph—S[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)OH —Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—OH—Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—NH₂ —Ph—S[(CH₂)₀₋₅CH₃]₂ Ph-Halo—Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—O(CH₂)₀₋₅CH₃ —Ph—S[(CH₂)₀₋₅CH₃]₂Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ —Ph—NO₂C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—NO₂ C(O)NHPh —Ph—NO₂ C(O)NH-2,4-fluorophenyl—Ph—NO₂ C(O)NH-4-fluorophenyl —Ph—NO₂ C(O)NH-3-fluorophenyl —Ph—NO₂C(O)NH-4-morpholinylphenyl —Ph—NO₂ C(O)NH-4-piperazinyllphenyl —Ph—NO₂C(O)NH-3-thiomethylphenyl —Ph—NO₂ H —Ph—NO₂ C(O)O(CH₂)₀₋₅CH₃ —Ph—NO₂C(O)OH —Ph—NO₂ Ph—OH —Ph—NO₂ Ph—NH₂ —Ph—NO₂ Ph-Halo —Ph—NO₂Ph—O(CH₂)₀₋₅CH₃ —Ph—NO₂ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—NO₂ C(O)NH₂ —Ph—CF₃C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—CF₃ C(O)NHPh —Ph—CF₃ C(O)NH-2,4-fluorophenyl—Ph—CF₃ C(O)NH-4-fluorophenyl —Ph—CF₃ C(O)NH-3-fluorophenyl —Ph—CF₃C(O)NH-4-morpholinylphenyl —Ph—CF₃ C(O)NH-4-piperazinyllphenyl —Ph—CF₃C(O)NH-3-thiomethylphenyl —Ph—CF₃ H —Ph—CF₃ C(O)O(CH₂)₀₋₅CH₃ —Ph—CF₃C(O)OH —Ph—CF₃ Ph—OH —Ph—CF₃ Ph—NH₂ —Ph—CF₃ Ph-Halo —Ph—CF₃Ph—O(CH₂)₀₋₅CH₃ —Ph—CF₃ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—CF₃ C(O)NH₂ NH₂,NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)N[(CH₂)₀₋₅CH₃]₂ NH₂,NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)NHPh NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ C(O)NH-2,4-fluorophenyl NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-fluorophenyl NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ C(O)NH-3-fluorophenyl NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-morpholinylphenyl NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-piperazinyllphenyl NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ C(O)NH-3-thiomethylphenyl NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ H NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)OH NH₂,NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—OH NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ Ph—NH₂ NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph-HaloNH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—O(CH₂)₀₋₅CH₃ NH₂,NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—N[(CH₂)₀₋₅CH₃]₂ NH₂, NH(CH₂)₀₋₅CH₃,or N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃C(O)N[(CH₂)₀₋₅CH₃]₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NHPh NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃C(O)NH-4-fluorophenyl NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenylNHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃H NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ C(O)OH NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph—OH NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ Ph—NH₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph-Halo NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃Ph—N[(CH₂)₀₋₅CH₃]₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH₂ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃C(O)NHPh NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHS(O)₂Hor NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃C(O)NH-4-morpholinylphenyl NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃C(O)NH-4-piperazinyllphenyl NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃C(O)NH-3-thiomethylphenyl NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ H NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃C(O)OH NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph—OH NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ Ph—NH₂ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph-HaloNHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃C(O)NH₂ OH or —O(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ OH or —O(CH₂)₀₋₅CH₃C(O)NHPh OH or —O(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl OH or —O(CH₂)₀₋₅CH₃C(O)NH-4-fluorophenyl OH or —O(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl OH or—O(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl OH or —O(CH₂)₀₋₅CH₃C(O)NH-4-piperazinyllphenyl OH or —O(CH₂)₀₋₅CH₃C(O)NH-3-thiomethylphenyl OH or —O(CH₂)₀₋₅CH₃ H OH or —O(CH₂)₀₋₅CH₃C(O)O(CH₂)₀₋₅CH₃ OH or —O(CH₂)₀₋₅CH₃ C(O)OH OH or —O(CH₂)₀₋₅CH₃ Ph—OH OHor —O(CH₂)₀₋₅CH₃ Ph—NH₂ OH or —O(CH₂)₀₋₅CH₃ Ph-Halo OH or —O(CH₂)₀₋₅CH₃Ph—O(CH₂)₀₋₅CH₃ OH or —O(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ OH or—O(CH₂)₀₋₅CH₃ C(O)NH₂ 1-pyrrolidyl C(O)N[(CH₂)₀₋₅CH₃]₂ 1-pyrrolidylC(O)NHPh 1-pyrrolidyl C(O)NH-2,4-fluorophenyl 1-pyrrolidylC(O)NH-4-fluorophenyl 1-pyrrolidyl C(O)NH-3-fluorophenyl 1-pyrrolidylC(O)NH-4-morpholinylphenyl 1-pyrrolidyl C(O)NH-4-piperazinyllphenyl1-pyrrolidyl C(O)NH-3-thiomethylphenyl 1-pyrrolidyl H 1-pyrrolidylC(O)O(CH₂)₀₋₅CH₃ 1-pyrrolidyl C(O)OH 1-pyrrolidyl Ph—OH 1-pyrrolidylPh—NH₂ 1-pyrrolidyl Ph-Halo 1-pyrrolidyl Ph—O(CH₂)₀₋₅CH₃ 1-pyrrolidylPh—N[(CH₂)₀₋₅CH₃]₂ 1-pyrrolidyl C(O)NH₂ 1-piperidinylC(O)N[(CH₂)₀₋₅CH₃]₂ 1-piperidinyl C(O)NHPh 1-piperidinylC(O)NH-2,4-fluorophenyl 1-piperidinyl C(O)NH-4-fluorophenyl1-piperidinyl C(O)NH-3-fluorophenyl 1-piperidinylC(O)NH-4-morpholinylphenyl 1-piperidinyl C(O)NH-4-piperazinyllphenyl1-piperidinyl C(O)NH-3-thiomethylphenyl 1-piperidinyl H 1-piperidinylC(O)O(CH₂)₀₋₅CH₃ 1-piperidinyl C(O)OH 1-piperidinyl Ph—OH 1-piperidinylPh—NH₂ 1-piperidinyl Ph-Halo 1-piperidinyl Ph—O(CH₂)₀₋₅CH₃ 1-piperidinylPh—N[(CH₂)₀₋₅CH₃]₂ 1-piperidinyl C(O)NH₂ 4-morpholinylC(O)N[(CH₂)₀₋₅CH₃]₂ 4-morpholinyl C(O)NHPh 4-morpholinylC(O)NH-2,4-fluorophenyl 4-morpholinyl C(O)NH-4-fluorophenyl4-morpholinyl C(O)NH-3-fluorophenyl 4-morpholinylC(O)NH-4-morpholinylphenyl 4-morpholinyl C(O)NH-4-piperazinyllphenyl4-morpholinyl C(O)NH-3-thiomethylphenyl 4-morpholinyl H 4-morpholinylC(O)O(CH₂)₀₋₅CH₃ 4-morpholinyl C(O)OH 4-morpholinyl Ph—OH 4-morpholinylPh—NH₂ 4-morpholinyl Ph-Halo 4-morpholinyl Ph—O(CH₂)₀₋₅CH₃ 4-morpholinylPh—N[(CH₂)₀₋₅CH₃]₂ 4-morpholinyl C(O)NH₂ —(CH₂)₀₋₅CH₃C(O)N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ C(O)NHPh —(CH₂)₀₋₅CH₃C(O)NH-2,4-fluorophenyl —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl —(CH₂)₀₋₅CH₃C(O)NH-3-fluorophenyl —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl—(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl —(CH₂)₀₋₅CH₃C(O)NH-3-thiomethylphenyl —(CH₂)₀₋₅CH₃ H —(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃—(CH₂)₀₋₅CH₃ C(O)OH —(CH₂)₀₋₅CH₃ Ph—OH —(CH₂)₀₋₅CH₃ Ph—NH₂ —(CH₂)₀₋₅CH₃Ph-Halo —(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ —(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂—(CH₂)₀₋₅CH₃ C(O)NH₂ H C(O)N[(CH₂)₀₋₅CH₃]₂ H C(O)NHPh HC(O)NH-2,4-fluorophenyl H C(O)NH-4-fluorophenyl H C(O)NH-3-fluorophenylH C(O)NH-4-morpholinylphenyl H C(O)NH-4-piperazinyllphenyl HC(O)NH-3-thiomethylphenyl H H H C(O)O(CH₂)₀₋₅CH₃ H C(O)OH H Ph—OH HPh—NH₂ H Ph-Halo H Ph—O(CH₂)₀₋₅CH₃ H Ph—N[(CH₂)₀₋₅CH₃]₂ H

TABLE 5

R¹ R² R³ pyrrolidinyl H, NO₂, CN, or SO₂CF₃ NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ piperidinyl H, NO₂, CN, or SO₂CF₃ NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ indole H, NO₂, CN, or SO₂CF₃ NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ NH₂ H, NO₂, CN, or SO₂CF₃ NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ —NHPh H, NO₂, CN, or SO₂CF₃ NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ Ph H, NO₂, CN, or SO₂CF₃ NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ OPh H, NO₂, CN, or SO₂CF₃ NH₂, NH(CH₂)₀₋₅CH₃, orN[(CH₂)₀₋₅CH₃]₂ pyrrolidinyl H, NO₂, CN, or SO₂CF₃ NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ piperidinyl H, NO₂, CN, or SO₂CF₃ NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ indole H, NO₂, CN, or SO₂CF₃ NHC(O)H orNHC(O)(CH₂)₀₋₅CH₃ NH₂ H, NO₂, CN, or SO₂CF₃ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃—NHPh H, NO₂, CN, or SO₂CF₃ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph H, NO₂, CN,or SO₂CF₃ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ OPh H, NO₂, CN, or SO₂CF₃ NHC(O)Hor NHC(O)(CH₂)₀₋₅CH₃ pyrrolidinyl H, NO₂, CN, or SO₂CF₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ piperidinyl H, NO₂, CN, or SO₂CF₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ indole H, NO₂, CN, or SO₂CF₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ NH₂ H, NO₂, CN, or SO₂CF₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ —NHPh H, NO₂, CN, or SO₂CF₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ Ph H, NO₂, CN, or SO₂CF₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ OPh H, NO₂, CN, or SO₂CF₃ NHS(O)₂H orNHS(O)₂(CH₂)₀₋₅CH₃ pyrrolidinyl H, NO₂, CN, or SO₂CF₃ H piperidinyl H,NO₂, CN, or SO₂CF₃ H indole H, NO₂, CN, or SO₂CF₃ H NH₂ H, NO₂, CN, orSO₂CF₃ H —NHPh H, NO₂, CN, or SO₂CF₃ H Ph H, NO₂, CN, or SO₂CF₃ H OPh H,NO₂, CN, or SO₂CF₃ H pyrrolidinyl H, NO₂, CN, or SO₂CF₃ —NHPhpiperidinyl H, NO₂, CN, or SO₂CF₃ —NHPh indole H, NO₂, CN, or SO₂CF₃—NHPh NH₂ H, NO₂, CN, or SO₂CF₃ —NHPh —NHPh H, NO₂, CN, or SO₂CF₃ —NHPhPh H, NO₂, CN, or SO₂CF₃ —NHPh OPh H, NO₂, CN, or SO₂CF₃ —NHPhpyrrolidinyl H, NO₂, CN, or SO₂CF₃ OH piperidinyl H, NO₂, CN, or SO₂CF₃OH indole H, NO₂, CN, or SO₂CF₃ OH NH₂ H, NO₂, CN, or SO₂CF₃ OH —NHPh H,NO₂, CN, or SO₂CF₃ OH Ph H, NO₂, CN, or SO₂CF₃ OH OPh H, NO₂, CN, orSO₂CF₃ OH pyrrolidinyl H, NO₂, CN, or SO₂CF₃ 1-pyrrolidyl piperidinyl H,NO₂, CN, or SO₂CF₃ 1-pyrrolidyl indole H, NO₂, CN, or SO₂CF₃1-pyrrolidyl NH₂ H, NO₂, CH, or SO₂CF₃ 1-pyrrolidyl —NHPh H, NO₂, CN, orSO₂CF₃ 1-pyrrolidyl Ph H, NO₂, CN, or SO₂CF₃ 1-pyrrolidyl OPh H, NO₂,CN, or SO₂CF₃ 1-pyrrolidyl pyrrolidinyl H, NO₂, CN, or SO₂CF₃1-piperidinyl piperidinyl H, NO₂, CN, or SO₂CF₃ 1-piperidinyl indole H,NO₂, CN, or SO₂CF₃ 1-piperidinyl NH₂ H, NO₂, CN, or SO₂CF₃ 1-piperidinyl—NHPh H, NO₂, CN, or SO₂CF₃ 1-piperidinyl Ph H, NO₂, CN, or SO₂CF₃1-piperidinyl OPh H, NO₂, CN, or SO₂CF₃ 1-piperidinyl pyrrolidinyl H,NO₂, CN, or SO₂CF₃ Ph piperidinyl H, NO₂, CN, or SO₂CF₃ Ph indole H,NO₂, CN, or SO₂CF₃ Ph NH₂ H, NO₂, CN, or SO₂CF₃ Ph —NHPh H, NO₂, CN, orSO₂CF₃ Ph Ph H, NO₂, CN, or SO₂CF₃ Ph OPh H, NO₂, CN, or SO₂CF₃ Phpyrrolidinyl H, NO₂, CN, or SO₂CF₃ —Ph-Halo piperidinyl H, NO₂, CN, orSO₂CF₃ —Ph-Halo indole H, NO₂, CN, or SO₂CF₃ —Ph-Halo NH₂ H, NO₂, CN, orSO₂CF₃ —Ph-Halo —NHPh H, NO₂, CN, or SO₂CF₃ —Ph-Halo Ph H, NO₂, CN, orSO₂CF₃ —Ph-Halo OPh H, NO₂, CN, or SO₂CF₃ —Ph-Halo

TABLE 6

R¹ R³ R² (Selected from one of the following) OC(O)NH₂ Halo C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ Halo C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH Halo C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ Halo C(O)NH₂₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃ Halo C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ Halo C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ Halo C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ Halo C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ OH C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ OH C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH OH C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ OH C(O)NH₂₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃ OH C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ OH C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ OH C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ OH C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ —O(CH₂)₀₋₅CH₃ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH —O(CH₂)₀₋₅CH₃C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂—O(CH₂)₀₋₅CH₃ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃OC(O)OC(O)C(CH)₃ —O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ —O(CH₂)₀₋₅CH₃ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ —O(CH₂)₀₋₅CH₃C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃—O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ NH₂C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ NH₂C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH NH₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ NH₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃ NH₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ NH₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ NH₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ NH₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OHN[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃OC(O)OP(O)(OH)₂ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ N[(CH₂)₀₋₅CH₃]₂C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃OC(O)(CH₂)₀₋₅CH₃ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ —(CH₂)₀₋₅CH₃ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH —(CH₂)₀₋₅CH₃ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(011)₂ —(CH₂)₀₋₅CH₃C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃—(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂—(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃OC(O)O(CH₂)₀₋₅CH₃ —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ —(CH₂)₀₋₅CH₃ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OHS[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃OC(O)OP(O)(OH)₂ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ S[(CH₂)₀₋₅CH₃]₂C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃OC(O)(CH₂)₀₋₅CH₃ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ NO₂ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OH NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ CF₃ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OH CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH)₃ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂C(O)O(CH₂)₀₋₅CH₃Certain embodiments can use any one or more of the following compounds:

III. Methods of Use

i. Methods of Treating Muscular Dystrophy

The α7β1 integrin has been shown to be a major modifier of diseaseprogression in patients with muscular dystrophy. Increased expression ofthe α7 integrin in muscle can alleviate muscle disease in mouse modelsof muscular dystrophy. By use of a muscle cell-based assay, theinventors identified molecules that up-regulate α7β1 integrin expressionin muscle: laminin-111; valproic acid; ciclopirox ethanolamine;deferoxamine; 2,2-dipyridyl; 5α-cholestan-3β-ol-6-one; Compound ID#1001;Compound ID#1002; Compound ID #1003; and analogs of cholestan. Based onthese observations, methods of treatment of muscular dystrophy byincreasing the expression or activity of α7β1 integrin with additionalsuitable compounds are disclosed.

In particular, methods are disclosed herein for treating musculardystrophy, such as DMD, FCMD, LGMD, FHMD, BMD, MDC1A or MDC1D. In oneexample, the method includes administering an effective amount of a α7β1integrin modulatory agent to a subject with muscular dystrophy orsuspected of having or developing muscular dystrophy, in which the agentincreases the biological activity or expression of α7β1 integrin andthereby, treating the muscular dystrophy in the subject. In someexample, the method of treatment inhibits or reduces one or more signsor symptoms associated with muscular dystrophy in the subject.

In some examples, the α7β1 integrin modulatory agent includes one ormore of the following molecules: a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18. In someexamples, an analog is synthesized according to the synthesis pathwayshown in the Schemes below. In further examples, the α7β1 integrinmodulatory agent is an analog/derivative of any of the disclosed α7β1integrin modulatory agents which may be designed and synthesizedaccording to the chemical principles known to one of ordinary skill inthe art and identified as a α7β1 integrin modulatory agent by methodsknown to those of ordinary skill in the art, including the muscle cellbased assay described Example 1. For examples, in some examples, theα7β1 integrin modulatory agent includes one or more molecules providedby any one of Formulas 1-16, or provided by any one of Tables 1-16 and18.

The disclosed α7β1 integrin modulatory agents can alter the expressionof nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins ofα7β1 integrin. An increase in the expression or activity does not needto be 100% for the agent to be effective. For example, an agent canincrease the expression or biological activity by a desired amount, forexample by at least 10%, for example at least 20%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or even at least 100%, including about 15% to about 98%,about 30% to about 95%, about 40% to about 80%, about 50% to about 70%,including about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, about 95%, about 98% or about 100%, ascompared to activity or expression in a control. Methods of assessingα7β1 integrin expression and activity are known to those of ordinaryskill in the art, including those described in the Examples below (e.g.,Western blot and ELISA assay with commercially available antibodies).

In a particular example, the subject is a human.

In additional aspects, the method involves selecting a subject withmuscular dystrophy. In some example, a subject is selected for treatmentfollowing diagnosing the subject with muscular dystrophy. For example,the method can include diagnosing the subject as suffering from musculardystrophy, such as DMD, MDC1A, MDC1D, LGMD, DMD, FCMD or FHMD.

Methods of diagnosing a subject with muscular dystrophy are known tothose of skill in the art and include, but are not limited to, musclebiopsies and measuring serum creatine kinase levels. Additionally,alterations in biomarker known to be associated with muscular dystrophymay be detected by measuring such levels in serum or urine sample.

In a further implementation, the method involves diagnosing the subjectas suffering from a disease, disorder, or condition characterized by amutation in the gene encoding α7 integrin. In another implementation,the method involves diagnosing the subject as suffering from a disease,disorder, or condition characterized by a decreased level of α7 integrinexpression.

Alterations in the expression can be measured at the nucleic acid level(such as by real time quantitative polymerase chain reaction ormicroarray analysis) or at the protein level (such as by Western blotanalysis or ELISA). These methods are known to those of skill in theart.

In some examples, following the measurement of the expression levels ofα7 integrin expression or serum creatine kinase levels, the assayresults, findings, diagnoses, predictions and/or treatmentrecommendations are recorded and communicated to technicians, physiciansand/or patients, for example. In certain embodiments, computers are usedto communicate such information to interested parties, such as, patientsand/or the attending physicians. The therapy selected for administeredis then based upon these results.

In one embodiment, the results and/or related information iscommunicated to the subject by the subject's treating physician.Alternatively, the results may be communicated directly to a testsubject by any means of communication, including writing, such as byproviding a written report, electronic forms of communication, such asemail, or telephone. Communication may be facilitated by use of acomputer, such as in case of email communications. In certainembodiments, the communication containing results of a diagnostic testand/or conclusions drawn from and/or treatment recommendations based onthe test, may be generated and delivered automatically to the subjectusing a combination of computer hardware and software which will befamiliar to artisans skilled in telecommunications. One example of ahealthcare-oriented communications system is described in U.S. Pat. No.6,283,761; however, the present disclosure is not limited to methodswhich utilize this particular communications system. In certainembodiments of the methods of the disclosure, all or some of the methodsteps, including the assaying of samples, diagnosing of diseases, andcommunicating of assay results or diagnoses, may be carried out indiverse (e.g., foreign) jurisdictions.

In several embodiments, identification of a subject as having musculardystrophy, such as DMD, LGMD, FHMD, BMD, FCMD, MDC1D or MDC1A, resultsin the physician treating the subject, such as prescribing one or moredisclosed α7β1 agents for inhibiting or delaying one or more signs andsymptoms associated with muscular dystrophy. In additional embodiments,the dose or dosing regimen is modified based on the information obtainedusing the methods disclosed herein.

ii. Methods of Enhancing Muscle Regeneration, Repair, or Maintenance

Also disclosed are methods of enhancing muscle regeneration, repair ormaintenance in a subject. In some examples, the method includesadministering an effective amount of an α7β1 integrin modulatory agentto a subject in need of muscle regeneration, repair or maintenance,wherein the α7β1 integrin modulatory agent includes a compoundencompassed by any one of Formulas 1-16, or provided by any one ofTables 1-16 and 18, or a combination thereof, wherein the α7β1 integrinmodulatory agent increases α7β1 integrin expression or activity ascompared to α7β1 integrin expression or activity prior to treatment,thereby enhancing muscle regeneration, repair or maintenance in asubject.

In some examples, the α7β1 integrin modulatory agent includes one ormore of the following molecules: a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18. In someexamples, an analog is synthesized according to the synthesis pathwayprovided in the Examples below. In further examples, the α7β1 integrinmodulatory agent is an analog/derivative of any of the disclosed α7β1integrin modulatory agents which may be designed and synthesizedaccording to the chemical principles known to one of ordinary skill inthe art and identified as a α7β1 integrin modulatory agent by methodsknown to those of ordinary skill in the art, including the muscle cellbased assay described Example 1. For examples, in some examples, theα7β1 integrin modulatory agent includes one or more molecules providedin a compound encompassed by any one of Formulas 1-16, or provided byany one of Tables 1-16 and 18.

The disclosed α7β1 integrin modulatory agents can increase theexpression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) andproteins of α7β1 integrin. An increase in the expression or activitydoes not need to be 100% for the agent to be effective. For example, anagent can increase the expression or biological activity by a desiredamount, for example by at least 10%, for example at least 20%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or even at least 100%, including about 15% to about98%, about 30% to about 95%, about 40% to about 80%, about 50% to about70%, including about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, ascompared to activity or expression in a control. Methods of assessingα7β1 integrin expression and activity are known to those of ordinaryskill in the art, including those described in the Examples below (e.g.,Western blot and ELISA assay with commercially available antibodies).

Muscle regeneration may benefit, for example, geriatric or other patientpopulations with reduced muscle repair capability, or simply speed themuscle repair process for otherwise physiologically unimpaired patients.In particular implementations, administration of a α7β1 integrinmodulatory agent can aid muscle repair, or reduction of muscle damage,in athletes or others having activity-induced muscle injury or damage.In yet further implementations, muscle repair in patients suffering frommuscle damage, such as through accident or injury, can be augmented byadministration of a α7β1 integrin modulatory agent.

In some examples, α7β1 modulatory agent is administered prior to thesubject experiencing muscle damage or disease. In some examples, theα7β1 integrin modulatory agent is administered to the subject prior tothe subject exercising.

In some examples, the method further includes selecting a subject inneed of enhancing muscle regeneration, repair, or maintenance. Forexample, in some instances, selecting a subject in need of enhancingmuscle regeneration, repair, or maintenance comprises diagnosing thesubject with a condition characterized by impaired muscle regenerationprior to administering an effective amount of the α7β1 integrinmodulatory agent to the subject. Methods for diagnosing and selecting asubject in need of muscle regeneration, repair or maintenance are knownto those of ordinary skill in the art and include those provideddescribed herein (including those in the Methods of Treatment ofMuscular Dystrophy). As stated above, subjects may be selected basedupon their life style (e.g., engaged in moderate to intense exercise orphysical activities), age (e.g., elderly population at more risk ofexperiencing muscle degeneration or injury) or pre-disposition to muscledegeneration or injury (e.g., genetics or previous muscle injury).

iii. Methods of Prospectively Preventing or Reducing Muscle Injury orDamage

Also disclosed are methods prospectively preventing or reducing muscleinjury or damage in a subject. In some embodiments, the method includesadministering an effective amount of an α7β1 integrin modulatory agentto the subject wherein the α7β1 integrin modulatory agent comprises acompound encompassed by any one of Formulas 1-16, or provided by any oneof Tables 1-16 and 18, or a combination thereof, wherein the α7β1integrin modulatory agent increases α7β1 integrin expression or activityas compared to α7β1 integrin expression or activity prior to treatment,thereby prospectively preventing or reducing muscle injury or damage inthe subject.

In some examples, the α7β1 integrin modulatory agent includes one ormore of the following molecules: a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18. In furtherexamples, the α7β1 integrin modulatory agent is an analog/derivative ofany of the disclosed α7β1 integrin modulatory agents which may bedesigned and synthesized according to the chemical principles known toone of ordinary skill in the art and identified as a α7β1 integrinmodulatory agent by methods known to those of ordinary skill in the art,including the muscle cell based assay described Example 1. For examples,in some examples, the α7β1 integrin modulatory agent includes one ormore molecules provided by any one of Formulas 1-16, or provided by anyone of Tables 1-16 and 18. An exemplary synthetic scheme for makingcertain α7β1 integrin modulatory agent disclosed herein is provided inSchemes 1 and 2 as illustrated in FIG. 12. A person of ordinary skill inthe art would recognize that derivatives of such compounds can beobtained using methods known in the art, such as functionalizing thecore structure using suitable reagents and conditions. Exemplary groupsthat can be modified to produce various analogs are indicated in theSchemes shown in FIG. 12; and in some embodiments, the core of themolecule can be modified to include one or more additional heteroatomsand/or to replace an existing heteroatom with a different suitableheteroatom.

The disclosed α7β1 integrin modulatory agents can increase theexpression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) andproteins of α7β1 integrin. An increase in the expression or activitydoes not need to be 100% for the agent to be effective. For example, anagent can increase the expression or biological activity by a desiredamount, for example by at least 10%, for example at least 20%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or even at least 100%, including about 15% to about98%, about 30% to about 95%, about 40% to about 80%, about 50% to about70%, including about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, ascompared to activity or expression in a control. Methods of assessingα7β1 integrin expression and activity are known to those of ordinaryskill in the art, including those described in the Examples below (e.g.,Western blot and ELISA assay with commercially available antibodies).

In some examples, the method further includes selecting a subject atrisk for developing a muscle injury or damage. In some examples, theα7β1 integrin modulatory agent is administered to a subject prior to thesubject exercising.

In some examples, the method further includes selecting a subject atrisk for developing a muscle injury or damage. Methods for selectingsuch s subject are known to those of ordinary skill in the art andinclude those provided described herein. As stated above, subjects maybe selected based upon their life style (e.g., engaged in moderate tointense exercise or physical activities), age (elderly population atmore risk of experiencing muscle degeneration or injury) orpre-disposition to muscle degeneration or injury (e.g., genetics orprevious muscle injury).

iv. Methods of Enhancing α7β1 Integrin Expression

Also disclosed herein are methods of enhancing α7β1 integrin expression.In some examples, these methods include contacting a cell with aneffective amount of an α7β1 integrin modulatory agent, wherein the α7β1integrin modulatory agent comprises a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18, or acombination thereof and increases α7β1 integrin expression in thetreated cell relative to α7β1 integrin expression in an untreated cell,thereby enhancing α7β1 integrin expression. In some examples, the cellis a muscle cell, such as a skeletal muscle cell. In some examples, themuscle cell is present in a mammal, and wherein contacting the cell withan agent comprises administering the agent to the mammal.

In some examples, the α7β1 integrin modulatory agent includes one ormore of the following molecules: a compound encompassed by any one ofFormulas 1-16, or provided by any one of Tables 1-16 and 18. In furtherexamples, the α7β1 integrin modulatory agent is an analog/derivative ofany of the disclosed α7β1 integrin modulatory agents which may bedesigned and synthesized according to the chemical principles known toone of ordinary skill in the art and identified as a α7β1 integrinmodulatory agent by methods known to those of ordinary skill in the art,including the muscle cell based assay described Example 1. In someexamples, the α7β1 integrin modulatory agent includes one or moremolecules encompassed by any one of Formulas 1-16, or provided by anyone of Tables 1-16 and 18.

In some examples, the disclosed α7β1 integrin modulatory agents canincrease the expression of nucleic acid sequences (such as DNA, cDNA, ormRNAs) and proteins of α7β1 integrin. An increase in the expression oractivity does not need to be 100% for the agent to be effective. Forexample, an agent can increase the expression or biological activity bya desired amount, for example by at least 10%, for example at least 20%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or even at least 100%, including about 15% toabout 98%, about 30% to about 95%, about 40% to about 80%, about 50% toabout 70%, including about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 95%, about 98% or about100%, as compared to activity or expression in a control. Methods ofassessing α7β1 integrin expression and activity are known to those ofordinary skill in the art, including those described in the Examplesbelow (e.g., Western blot and ELISA assay with commercially availableantibodies).

Administration of an Effective Amount of an α7β1 Integrin ModulatoryAgent

For any of the disclosed methods, an effective amount of α7β1 integrinmodulatory agent is one when administered by a particular route andconcentration induces the desired response (e.g., treatment of musculardystrophy, enhancing muscle regeneration, repair or maintenance,preventing or reducing muscle injury or damage, or enhancing α7β1integrin expression).

i. Administration Routes, Formulations and Concentrations

Methods of administration of the disclosed α7β1 integrin modulatoryagents are routine, and can be determined by a skilled clinician. Thedisclosed α7β1 integrin modulatory agents or other therapeutic substanceare in general administered topically, nasally, intravenously, orally,intracranially, intramuscularly, parenterally or as implants, but evenrectal or vaginal use is possible in principle. The disclosed α7β1integrin modulatory agents also may be administered to a subject using acombination of these techniques.

Suitable solid or liquid pharmaceutical preparation forms are, forexample, aerosols, (micro)capsules, creams, drops, drops or injectablesolution in ampoule form, emulsions, granules, powders, suppositories,suspensions, syrups, tablets, coated tablets, and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as binders, coating agents,disintegrants, flavorings, lubricants, solubilizers, sweeteners, orswelling agents are customarily used as described above. Thepharmaceutical agents are suitable for use in a variety of drug deliverysystems. For a brief review of various methods for drug delivery, seeLanger, “New Methods of Drug Delivery,” Science 249:1527-1533 (1990),incorporated by reference herein to the extent not inconsistent with thepresent disclosure.

The disclosed α7β1 integrin modulatory agents or other therapeuticagents of the present disclosure can be formulated intotherapeutically-active pharmaceutical agents that can be administered toa subject parenterally or orally. Parenteral administration routesinclude, but are not limited to epidermal, intraarterial, intramuscular(IM and depot IM), intraperitoneal (IP), intravenous (IV), intrasternalinjection or infusion techniques, intranasal (inhalation), intrathecal,injection into the stomach, subcutaneous injections (subcutaneous (SQand depot SQ), transdermal, topical, and ophthalmic.

The disclosed α7β1 integrin modulatory agents or other therapeuticagents can be mixed or combined with a suitable pharmaceuticallyacceptable excipients to prepare pharmaceutical agents. Pharmaceuticallyacceptable excipients include, but are not limited to, alumina, aluminumstearate, buffers (such as phosphates), glycine, ion exchangers (such asto help control release of charged substances), lecithin, partialglyceride mixtures of saturated vegetable fatty acids, potassiumsorbate, serum proteins (such as human serum albumin), sorbic acid,water, salts or electrolytes such as cellulose-based substances,colloidal silica, disodium hydrogen phosphate, magnesium trisilicate,polyacrylates, polyalkylene glycols, such as polyethylene glycol,polyethylene-polyoxypropylene-block polymers, polyvinyl pyrrolidone,potassium hydrogen phosphate, protamine sulfate, group 1 halide saltssuch as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat,and zinc salts, for example. Liposomal suspensions may also be suitableas pharmaceutically acceptable carriers.

Upon mixing or addition of one or more disclosed α7β1 integrinmodulatory agents and/or or other therapeutic agents, the resultingmixture may be a solid, solution, suspension, emulsion, or the like.These may be prepared according to methods known to those of ordinaryskill in the art. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the agent in the selected carrier. Pharmaceutical carrierssuitable for administration of the disclosed α7β1 integrin modulatoryagents or other therapeutic agents include any such carriers known to besuitable for the particular mode of administration. In addition, thedisclosed α7β1 integrin modulatory agents or other therapeutic substancecan also be mixed with other inactive or active materials that do notimpair the desired action, or with materials that supplement the desiredaction, or have another action.

Methods for solubilizing may be used where the agents exhibitinsufficient solubility in a carrier. Such methods are known andinclude, but are not limited to, dissolution in aqueous sodiumbicarbonate, using cosolvents such as dimethylsulfoxide (DMSO), andusing surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, Del.).

The disclosed α7β1 integrin modulatory agents or other therapeuticagents can be prepared with carriers that protect them against rapidelimination from the body, such as coatings or time-releaseformulations. Such carriers include controlled release formulations,such as, but not limited to, microencapsulated delivery systems. Adisclosed α7β1 integrin modulatory agents or other therapeutic agent isincluded in the pharmaceutically acceptable carrier in an amountsufficient to exert a therapeutically useful effect, typically in anamount to avoid undesired side effects, on the treated subject. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in known in vitro and in vivo model systems forthe treated condition. For example, mouse models of muscular dystrophymay be used to determine effective amounts or concentrations that canthen be translated to other subjects, such as humans, as known in theart.

Injectable solutions or suspensions can be formulated, using suitablenon-toxic, parenterally-acceptable diluents or solvents, such as1,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer'ssolution, saline solution, or water; or suitable dispersing or wettingand suspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid;a naturally occurring vegetable oil such as coconut oil, cottonseed oil,peanut oil, sesame oil, and the like; glycerine; polyethylene glycol;propylene glycol; or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfate; buffers such as acetates, citrates, andphosphates; chelating agents such as ethylenediaminetetraacetic acid(EDTA); agents for the adjustment of tonicity such as sodium chlorideand dextrose; and combinations thereof. Parenteral preparations can beenclosed in ampoules, disposable syringes, or multiple dose vials madeof glass, plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required. Whereadministered intravenously, suitable carriers include physiologicalsaline, phosphate-buffered saline (PBS), and solutions containingthickening and solubilizing agents such as glucose, polyethylene glycol,polypropyleneglycol, and mixtures thereof. Liposomal suspensions,including tissue-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers.

For topical application, one or more disclosed α7β1 integrin modulatoryagents, or other therapeutic agent may be made up into a cream, lotion,ointment, solution, or suspension in a suitable aqueous or non-aqueouscarrier. Topical application can also be accomplished by transdermalpatches or bandages which include the therapeutic substance. Additivescan also be included, e.g., buffers such as sodium metabisulphite ordisodium edetate; preservatives such as bactericidal and fungicidalagents, including phenyl mercuric acetate or nitrate, benzalkoniumchloride, or chlorhexidine; and thickening agents, such as hypromellose.

If the disclosed α7β1 integrin modulatory agent, or other therapeuticagent is administered orally as a suspension, the pharmaceutical agentscan be prepared according to techniques well known in the art ofpharmaceutical formulation and may contain a suspending agent, such asalginic acid or sodium alginate, bulking agent, such as microcrystallinecellulose, a viscosity enhancer, such as methylcellulose, andsweeteners/flavoring agents. Oral liquid preparations can containconventional additives such as suspending agents, e.g., gelatin, glucosesyrup, hydrogenated edible fats, methyl cellulose, sorbitol, and syrup;emulsifying agents, e.g., acacia, lecithin, or sorbitan monooleate;non-aqueous carriers (including edible oils), e.g., almond oil,fractionated coconut oil, oily esters such as glycerine, propyleneglycol, or ethyl alcohol; preservatives such as methyl or propylp-hydroxybenzoate or sorbic acid; and, if desired, conventionalflavoring or coloring agents. When formulated as immediate releasetablets, these agents can contain dicalcium phosphate, lactose,magnesium stearate, microcrystalline cellulose, and starch and/or otherbinders, diluents, disintegrants, excipients, extenders, and lubricants.

If oral administration is desired, one or more disclosed α7β1 integrinmodulatory agents, or other therapeutic substances can be provided in acomposition that protects it from the acidic environment of the stomach.For example, he disclosed α7β1 integrin modulatory agents or othertherapeutic agents can be formulated with an enteric coating thatmaintains its integrity in the stomach and releases the active compoundin the intestine. The disclosed α7β1 integrin modulatory agents, orother therapeutic agent can also be formulated in combination with anantacid or other such ingredient.

Oral compositions generally include an inert diluent or an ediblecarrier and can be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, one ormore of the disclosed α7β1 integrin modulatory agents, or othertherapeutic substances can be incorporated with excipients and used inthe form of capsules, tablets, or troches. Pharmaceutically compatibleadjuvant materials or binding agents can be included as part of thecomposition.

The capsules, pills, tablets, troches, and the like can contain any ofthe following ingredients or compounds of a similar nature: a bindersuch as, but not limited to, acacia, corn starch, gelatin, gumtragacanth, polyvinylpyrrolidone, or sorbitol; a filler such as calciumphosphate, glycine, lactose, microcrystalline cellulose, or starch; adisintegrating agent such as, but not limited to, alginic acid and cornstarch; a lubricant such as, but not limited to, magnesium stearate,polyethylene glycol, silica, or talc; a gildant, such as, but notlimited to, colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; disintegrants such as potato starch; dispersing orwetting agents such as sodium lauryl sulfate; and a flavoring agent suchas peppermint, methyl salicylate, or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier, such as a fatty oil. Inaddition, dosage unit forms can contain various other materials thatmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. One or more of the disclosed α7β1integrin modulatory agents, or other therapeutic agent can also beadministered as a component of an elixir, suspension, syrup, wafer, tea,chewing gum, or the like. A syrup may contain, in addition to the activecompounds, sucrose or glycerin as a sweetening agent and certainpreservatives, dyes and colorings, and flavors.

When administered orally, the compounds can be administered in usualdosage forms for oral administration. These dosage forms include theusual solid unit dosage forms of tablets and capsules as well as liquiddosage forms such as solutions, suspensions, and elixirs. When the soliddosage forms are used, they can be of the sustained release type so thatthe compounds need to be administered less frequently.

In some examples, one or more of the disclosed α7β1 integrin modulatoryagents and/or a therapeutic agent is injected into the stomach of asubject is incorporated systemically in the subject, such as in diversemuscle groups. Examples of methods and compositions for administeringtherapeutic substances which include proteins include those discussed inBanga, Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems 2ed. (2005); Mahato, Biomaterials for Delivery andTargeting of Proteins and Nucleic Acids (2004); McNally, ProteinFormulation and Delivery, 2ed. (2007); and Kumar et al., “Novel DeliveryTechnologies for Protein and Peptide Therapeutics,” Current Pharm.Biotech., 7:261-276 (2006); each of which is incorporated by referenceherein to the extent not inconsistent with the present disclosure.

In some implementations, the effective amount of one or more of thedisclosed α7β1 integrin modulatory agents is administered as a singledose per time period, such as every three or four months, month, week,or day, or it can be divided into at least two unit dosages foradministration over a period. Treatment may be continued as long asnecessary to achieve the desired results. For instance, treatment maycontinue for about 3 or 4 weeks up to about 12-24 months or longer,including ongoing treatment. The compound can also be administered inseveral doses intermittently, such as every few days (for example, atleast about every two, three, four, five, or ten days) or every fewweeks (for example at least about every two, three, four, five, or tenweeks).

Particular dosage regimens can be tailored to a particular subject,condition to be treated, or desired result. For example, when themethods of the present disclosure are used to treat muscular dystrophyor similar conditions, an initial treatment regimen can be applied toarrest the condition. Such initial treatment regimen may includeadministering a higher dosage of one or more of the disclosed α7β1integrin modulatory agents, or administering such material morefrequently, such as daily. After a desired therapeutic result has beenobtained, such as a desired level of muscle regeneration, a secondtreatment regimen may be applied, such as administering a lower dosageof one or more of the disclosed α7β1 integrin modulatory agents oradministering such material less frequently, such as monthly,bi-monthly, quarterly, or semi-annually. In such cases, the secondregimen may serve as a “booster” to restore or maintain a desired levelof muscle regeneration. Similar treatment regimens may be used for othersubjects with reduced or impaired muscle regeneration capabilities, suchas geriatric subjects.

When particular methods of the present disclosure are used to prevent ormitigate muscle damage, such as damage caused by exertion or injury, thesubject is typically treated a sufficient period of time before theexertion or injury in order to provide therapeutic effect. For example,the subject may be treated at least about 24 hours before the expectedactivity or potential injury, such as at least about 48 hours, about 72hours, about 1 week, about 2 weeks, about three weeks, or about 4 weeksor more prior.

When embodiments of the method of the present disclosure are used toprevent or treat a muscle injury, one or more of the disclosed α7β1integrin modulatory agents or other therapeutic substance can be applieddirectly to, or proximately to, the area to be treated. For example, thesubstance can be injected into or near the area. In further examples,the substance can be applied topically to the area to be treated.Treatment is typically initiated prior to the injury to several weeksfollowing the injury. In more specific implementations, the treatment isinitiated between about 12 and about 72 hours following injury, such asbetween about 24 and about 48 hours following injury. In some cases, asingle administration of the substance is effective to provide thedesired therapeutic effect. In further examples, additionaladministrations are provided in order to achieve the desired therapeuticeffect.

Amounts effective for various therapeutic treatments of the presentdisclosure may, of course, depend on the severity of the disease and theweight and general state of the subject, as well as the absorption,inactivation, and excretion rates of the therapeutically-active compoundor component, the dosage schedule, and amount administered, as well asother factors known to those of ordinary skill in the art. It alsoshould be apparent to one of ordinary skill in the art that the exactdosage and frequency of administration will depend on the particularα7β1 integrin modulatory agent, or other therapeutic substance beingadministered, the particular condition being treated, the severity ofthe condition being treated, the age, weight, general physical conditionof the particular subject, and other medication the subject may betaking. Typically, dosages used in vitro may provide useful guidance inthe amounts useful for in vivo administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of particular disorders. For example, mouse modelsof muscular dystrophy may be used to determine effective dosages thatcan then be translated to dosage amount for other subjects, such ashumans, as known in the art. Various considerations in dosagedetermination are described, e.g., in Gilman et al., eds., Goodman AndGilman's: The Pharmacological Bases of Therapeutics, 8th ed., PergamonPress (1990); and Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Co., Easton, Pa. (1990), each of which is herein incorporatedby reference to the extent not inconsistent with the present disclosure.

In specific examples, the one or more disclosed α7β1 integrin modulatoryagents is administered to a subject in an amount sufficient to provide adose of the agent of between about 10 fmol/g and about 500 nmol/g, suchas between about 2 nmol/g and about 20 nmol/g or between about 2 nmol/gand about 10 nmol/g. In additional examples, the α7β1 integrinmodulatory agent is administered to a subject in an amount sufficient toprovide a dose of between about 0.01 μg/kg and about 1000 mg/kg orbetween about 0.1 mg/kg and about 1000 mg/kg, in particular examplesthis amount is provided per day or per week. In another example, thedisclosed α7β1 integrin modulatory agent is administered to a subject inan amount sufficient to provide a dose of agent of between about 0.2mg/kg and about 2 mg/kg. In further examples, the α7β1 integrinmodulatory agent is administered to a subject in an amount sufficient toprovide a concentration of α7β1 integrin modulatory agent in theadministrated material of between about 5 nM and about 500 nM, such asbetween about 50 nM and about 200 nm, or about 100 nM. In otherexamples, the α7β1 integrin modulatory agent is administered to asubject between about 500 μg/ml and about 1 μg/ml, such as about 300μg/ml and about 3 μg/ml, about 200 μg/ml and about 20 μg/ml, including500 μg/ml, 400 μg/ml, 300 μg/ml, 250 μg/ml, 200 μg/ml, 150 μg/ml, 100μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.125 μg/ml, 2.5μg/ml and 1.25 μg/ml.

ii. Desired Response

One or more disclosed α7β1 integrin modulatory agents and/or additionaltherapeutic agents are administered by a specific route and/orconcentration to generate a desired response. In some examples, adesired response refers to an amount effective for lessening,ameliorating, eliminating, preventing, or inhibiting at least onesymptom of a disease, disorder, or condition treated and may beempirically determined. In various embodiments of the presentdisclosure, a desired response is muscle regeneration, reductions orprevention of muscle degeneration, promotion of muscle maintenance,reduction or prevention of muscle injury or damage, reduction orprevention in one more signs or symptoms associated with musculardystrophy.

In particular, indicators of muscular health, such as muscle cellregeneration, maintenance, or repair, can be assessed through variousmeans, including monitoring markers of muscle regeneration, such astranscription factors such as Pax7, Pax3, MyoD, MRF4, and myogenin. Forexample, increased expression of such markers can indicate that muscleregeneration is occurring or has recently occurred. Markers of muscleregeneration, such as expression of embryonic myosin heavy chain(eMyHC), can also be used to gauge the extent of muscle regeneration,maintenance, or repair. For example, the presence of eMyHC can indicatethat muscle regeneration has recently occurred in a subject.

Muscle cell regeneration, maintenance, or repair can also be monitoredby determining the girth, or mean cross sectional area, of muscle cellsor density of muscle fibers. Additional indicators of muscle conditioninclude muscle weight and muscle protein content. Mitotic index (such asby measuring BrdU incorporation) and myogenesis can also be used toevaluate the extent of muscle regeneration.

In particular examples, the improvement in muscle condition, such asregeneration, compared with a control is at least about 10%, such as atleast about 30%, or at least about 50% or more, including an at least15%, at least 20%, at least 25%, at least 30%, at least 40%, at least45%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, including a 10% to 90% decrease, 20% to 80% increase, 30% to 70%increase or a 40% to 60% increase (e.g., a 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 100%, 200% or more increase).

iii. Additional Treatments or Therapeutic Agents

In particular examples, prior to, during, or following administration ofan effective amount of an agent that reduces or inhibits one or moresigns or symptoms associated with muscular dystrophy, the subject canreceive one or more other therapies. In one example, the subjectreceives one or more treatments prior to administration of a disclosedα7β1 modulatory agent. Examples of such therapies include, but are notlimited to, laminin-111 protein therapy, which works to stabilize thesarcolemma and reduce muscle degeneration. In some examples, a source ofmuscle cells can be added to aid in muscle regeneration and repair. Insome aspects of the present disclosure, satellite cells are administeredto a subject in combination with laminin therapy. U.S. PatentPublication 2006/0014287, incorporated by reference herein to the extentnot inconsistent with the present disclosure, provides methods ofenriching a collection of cells in myogenic cells and administeringthose cells to a subject. In further aspects, stem cells, such asadipose-derived stem cells, are administered to the subject. Suitablemethods of preparing and administering adipose-derived stem cells aredisclosed in U.S. Patent Publication 2007/0025972, incorporated byreference herein to the extent not inconsistent with the presentdisclosure. Additional cellular materials, such as fibroblasts, can alsobe administered, in some examples.

Additional therapeutic agents include agents which enhance the effect ofthe disclosed α7β1 modulatory agents, such as a component of theextracellular matrix, such as an integrin, dystrophin, dystroglycan,utrophin, or a growth factor. In some examples, the additionaltherapeutic agent reduces or enhances expression of a substance thatenhances the formation or maintenance of the extracellular matrix. Insome examples, the additional substance can include aggrecan,angiostatin, cadherins, collagens (including collagen I, collagen III,or collagen IV), decorin, elastin, enactin, endostatin, fibrin,fibronectin, osteopontin, tenascin, thrombospondin, vitronectin, andcombinations thereof. Biglycans, glycosaminoglycans (such as heparin),glycoproteins (such as dystroglycan), proteoglycans (such as heparansulfate), and combinations thereof can also be administered.

In some examples, growth stimulants such as cytokines, polypeptides, andgrowth factors such as brain-derived neurotrophic factor (BDNF), CNF(ciliary neurotrophic factor), EGF (epidermal growth factor), FGF(fibroblast growth factor), glial growth factor (GGF), glial maturationfactor (GMF) glial-derived neurotrophic factor (GDNF), hepatocyte growthfactor (HGF), insulin, insulin-like growth factors, kerotinocyte growthfactor (KGF), nerve growth factor (NGF), neurotropin-3 and -4, PDGF(platelet-derived growth factor), vascular endothelial growth factor(VEGF), and combinations thereof may be administered with one of thedisclosed methods.

IV. Clinical Trials

To obtain regulatory approval for the use of one or more of thedisclosed α7β1 modulatory agents to treat a muscular disorder, clinicaltrials are performed. As is known in the art, clinical trials progressthrough phases of testing, which are identified as Phases I, II, III,and IV.

Initially the disclosed α7β1 modulatory agent is evaluated in a Phase Itrial. Typically Phase I trials are used to determine the best mode ofadministration (for example, by pill or by injection), the frequency ofadministration, and the toxicity for the compounds. Phase I studiesfrequently include laboratory tests, such as blood tests and biopsies,to evaluate the effects of the potential therapeutic in the body of thepatient. For a Phase I trial, a small group of patients with a musculardisorder are treated with a specific dose of a disclosed α7β1 modulatoryagent. During the trial, the dose is typically increased group by groupin order to determine the maximum tolerated dose (MTD) and thedose-limiting toxicities (DLT) associated with the compound. Thisprocess determines an appropriate dose to use in a subsequent Phase IItrial.

A Phase II trial can be conducted to further evaluate the effectivenessand safety of the disclosed α7β1 modulatory agent. In Phase II trials, adisclosed α7β1 modulatory agent is administered to groups of patientswith a muscular disorder using the dosage found to be effective in PhaseI trials.

Phase III trials focus on determining how a disclosed α7β1 modulatoryagent compares to the standard, or most widely accepted, treatment. InPhase III trials, patients are randomly assigned to one of two or more“arms”. In a trial with two arms, for example, one arm will receive thestandard treatment (control group) and the other arm will receive adisclosed α7β1 modulatory agent treatment (investigational group).

Phase IV trials are used to further evaluate the long-term safety andeffectiveness of a disclosed α7β1 modulatory agent. Phase IV trials areless common than Phase I, II and III trials and take place after adisclosed α7β1 modulatory agent has been approved for standard use.

Eligibility of Patients for Clinical Trials

Participant eligibility criteria can range from general (for example,age, sex, type of disease) to specific (for example, type and number ofprior treatments, disease characteristics, blood cell counts, organfunction). In one embodiment, eligible patients have been diagnosed witha muscular disorder. Eligibility criteria may also vary with trialphase. Patients eligible for clinical trials can also be chosen based onobjective measurement of a muscular disorder and failure to respond toother muscular disorder treatments. For example, in Phase I and IItrials, the criteria often exclude patients who may be at risk from theinvestigational treatment because of abnormal organ function or otherfactors. In Phase II and III trials additional criteria are oftenincluded regarding disease type and stage, and number and type of priortreatments.

Phase I trials usually include 15 to 30 participants for whom othertreatment options have not been effective. Phase II trials typicallyinclude up to 100 participants who have already received drug therapy,but for whom the treatment has not been effective.

Participation in Phase III trials is often restricted based on theprevious treatment received. Phase III trials usually include hundredsto thousands of participants. This large number of participants isnecessary in order to determine whether there are true differencesbetween the effectiveness of a disclosed α7β1 modulatory agent and thestandard treatment. Phase III can include patients ranging from thosenewly diagnosed with a muscular disorder to those with re-occurringsigns and/or symptoms associated with a muscular disorder or a musculardisorder that did not respond to prior treatment.

One skilled in the art will appreciate that clinical trials should bedesigned to be as inclusive as possible without making the studypopulation too diverse to determine whether the treatment might be aseffective on a more narrowly defined population. The more diverse thepopulation included in the trial, the more applicable the results couldbe to the general population, particularly in Phase III trials.Selection of appropriate participants in each phase of clinical trial isconsidered to be within the ordinary skills of a worker in the art.

Assessment of Patients Prior to Treatment

Prior to commencement of the study, several measures known in the artcan be used to first classify the patients. Patients can first beassessed, for example by determining serum creatine kinase (CK) levelsor other indicators of a muscle disorder, such as increased levels ofmuscle inflammation, apoptosis, muscle loss, myotube hypertrophy, and/ordecreased myofibers stability and cell survival.

Administration of a Disclosed α7β1 Modulatory Agent in Clinical Trials

A disclosed α7β1 modulatory agent is typically administered to the trialparticipants orally. A range of doses of the agent can be tested.Provided with information from preclinical testing, a skilledpractitioner can readily determine appropriate dosages of agent for usein clinical trials. In one embodiment, a dose range is from about 100μg/kg and about 5000 mg/kg of the subject's weight, such as 1 mg/kg andabout 2000 mg/kg of the subject's weight, about 100 mg/kg and about 1500mg/kg of the subject's weight, about 100 μg/kg and about 2000 mg/kg ofthe subject's weight, about 200 mg/kg and about 1000 mg/kg of thesubject's weight, about 200 mg/kg and about 750 mg/kg of the subject'sweight, about 250 mg/kg and about 500 mg/kg of the subject's weight,about 100 μm and about 500 mM. In some embodiments, subjects are given adisclosed α7β1 modulatory agent orally at 10 to 60 mg/kg of body weightper day. For example, 10-15 mg/kg of a disclosed α7β1 modulatory agentis administered for two weeks and if well tolerated the dose isincreased by 5-10 mg/kg/week to achieve optimal clinical response. Insome examples, the daily dose does not exceed 60 mg/kg of body weightand is given for a minimum of 6 months with liver function monitoredevery two weeks to monthly.

Pharmacokinetic Monitoring

To fulfill Phase I criteria, distribution of the disclosed α7β1modulatory agent is monitored, for example, by chemical analysis ofsamples, such as blood, collected at regular intervals. For example,samples can be taken at regular intervals up until about 72 hours afterthe start of treatment.

If analysis is not conducted immediately, the samples can be placed ondry ice after collection and subsequently transported to a freezer to bestored at −70° C. until analysis can be conducted. Samples can beprepared for analysis using standard techniques known in the art and theamount of the disclosed α7β1 modulatory agent present can be determined,for example, by high-performance liquid chromatography (HPLC).Pharmacokinetic data can be generated and analyzed in collaboration withan expert clinical pharmacologist and used to determine, for example,clearance, half-life and maximum plasma concentration.

Monitoring of Patient Outcome

The endpoint of a clinical trial is a measurable outcome that indicatesthe effectiveness of a compound under evaluation. The endpoint isestablished prior to the commencement of the trial and will varydepending on the type and phase of the clinical trial. Examples ofendpoints include, for example, decline in serum CK levels,inflammation, apoptosis, and muscle loss. For example, at least a 10%reduction in serum CK levels indicates the patient is responsive to thetreatment.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1

This example demonstrates the results of quantitative real-time PCR usedto assess Itga7, Itgb1, and Lama2 transcript levels in C2C12 myoblastsand myotubes treated for 24 hours with DMSO control, 10 μMMLS000683232-01 (IED-232), 10 μM MLS001165937-01 (IED-937),Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 inHPBCD (FIG. 1). FIG. 2 is a digital image of Western Blots andquantitative analysis of α7 Integrin and GAPDH protein levels in C2C12myotubes treated for 48 hours with DMSO control, 10 μM MLS000683232-01(IED-232), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μMSU9516 in HPBCD. Bands were quantified using Image J software and thengraphed as α7 Integrin protein levels relative to GAPDH proteinlevels. * denotes a significant difference in relative protein levelswith ** p<0.01.

Example 2

The additional structures provided below provide additional compoundsthat may be used in the methods disclosed herein increasing α7 integrinexpression in muscle (see Table 7). In some examples, the analogs weremade in 5 mg quantities, salt form (e.g., hydrochloride salt), as a drypowder, at an at least 90% purity as measured by HPLC. In someembodiments, physical properties (kinetic, solubility, PAMPApermeability, clog P) and metabolic stability (plasma and microsomalstability, metabolite identification) of all synthesized molecules, aswell as measure pharmacokinetics properties, compound levels anddistribution of selected compounds are determined. Besides systematicsubstitution for exploring selected chemotypes, classical medicinalchemistry parameters (MW, number of hydrogen donors and acceptors, tPSA,clogP, flexibility of the molecule) as well as in house-measuredphysical and metabolic properties of analog molecules are used to guidethe selection of potent, efficacious and non-toxic compounds havingsuitable pharmacokinetic properties for use in the disclosed methods.

TABLE 7 Exemplary Compounds

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

Compound 35

Compound 36

Compound 37

Compound 38

Compound 39

Compound 40

Compound 41

Compound 42

Compound 43

Compound 44

Compound 45

Compound 46

Compound 47

Compound 48

Compound 49

Compound 50

Compound 51

Compound 52

Compound 53

Compound 54

Compound 55

Compound 56

Compound 57

Compound 58

Compound 59

Compound 60

Compound 61

Compound 62

Compound 63

Compound 64

Compound 65

Compound 66

Compound 67

Compound 68

Compound 69

Compound 70

Compound 71

Compound 72

Compound 73

Compound 74

Compound 75

Compound 76

Compound 77

Compound 78

Compound 79

Compound 80

Compound 81

Compound 82

Compound 83

Compound 84

Compound 85

Compound 86

Compound 87

Compound 88

Compound 89

Compound 90

Compound 91

Compound 92

Compound 93

Compound 94

Compound 95

Compound 96

Compound 97

Compound 98

Compound 99

Compound 100

Compound 101

Compound 102

Compound 103

Compound 104

Compound 105

Compound 106

Compound 107

Compound 108

Compound 109

Compound 110

Compound 111

Compound 112

Compound 113

Compound 114

Compound 115

Compound 116

Compound 117

Compound 118

Compound 119

Compound 120

Compound 121

Compound 122

Compound 123

Compound 124

Compound 125

Compound 126

Compound 127

Compound 128

Compound 129

Compound 130

Compound 131

Compound 132

Compound 133

Compound 134

Compound 135

Compound 136

Compound 137

Compound 138

Compound 139

Compound 140

Compound 141

Compound 142

Compound 143

Compound 144

Compound 145

Compound 146

Compound 147

Compound 148

Compound 149

Compound 150

Compound 151

Compound 152

Compound 153

Compound 154

Compound 155

Compound 156

Compound 157

Compound 158

Compound 159

Compound 160

Compound 161

Compound 162

Compound 163

Compound 164

Compound 165

Compound 166

Compound 167

Compound 168

Compound 169

Compound 170

Compound 171

Compound 172

Compound 173

Compound 174

Compound 175

Compound 176

Compound 177

Compound 178

Compound 179

Compound 180

Compound 181

Compound 182

Compound 183

Compound 184

Compound 185

Compound 186

Compound 187

Compound 188

Compound 189

Compound 190

Compound 191

Compound 192

Compound 193

Compound 194

Compound 195

Compound 196

Compound 197

Compound 198

Compound 199

Compound 200

Compound 201

Compound 202

Compound 203

Compound 204

Compound 205

Compound 206

Compound 207

Compound 208

Compound 209

Compound 210

Compound 211

Compound 212

Compound 213

Compound 214

Compound 215

Compound 216

Compound 217

Compound 218

Compound 219

Compound 220

Compound 221

Compound 222

TABLE 8 Compound data Fit Fit Fit Infinite Zero Fit Max Activity atActivity at Activity at Activity at Curve LogA Fit Hill ActivityActivity Curve Excl'd Response 0.307 uM 1.530 uM 7.660 uM 38.30uM No.Score Description C50 Slope Fit R2 [%] [%] Class Points [%] [%] ** [%]** [%] ** [%] ** 1 94 Complete −5.95 3.132 0.9985 195.171 −2.9752 1.1 00 0 0 190.551 0 142.678 198.344 190.551 curve; high efficacy 2 91Complete −5.45 1.4163 0.9999 231.285 3.1995 1.1 0 0 0 0 222.818 9.519956.9884 175.202 222.818 curve; high efficacy 3 89 Complete −5.65 3.06540.9999 158.124 −35.3084 1.1 0 0 0 0 158.59 −34.2446 9.5695 153.966158.59 curve; high efficacy 4 87 Complete −5.55 1.8617 0.9998 136.9057.6069 1.1 0 0 0 0 136.632 9.8511 38.314 118.549 136.632 curve; highefficacy 5 86 Complete −5.95 2.5334 0.9999 87.4201 −5.0637 1.1 0 0 0 087.1241 −1.7993 58.4455 87.2456 687.1241 curve; high efficacy 6 85Complete −5.95 1.3437 0.9999 69.589 −58.5311 1.1 0 0 0 0 68.1048−39.7851 19.1903 60.5602 68.1048 curve; high efficacy 7 85 Complete−5.35 1.6924 0.9999 107.421 12.5 1.1 0 0 0 0 104.699 13.4132 25.272780.6487 104.699 curve; high efficacy 8 85 Complete −5.55 4.9549 0.999696.0327 −15.6523 1.1 0 0 0 0 94.3383 −15.3725 −10.6082 96.4184 94.3383curve; high efficacy 9 85 Complete −5.55 1.3723 0.9999 92.2022 −30.50051.1 0 0 0 0 88.6572 −24.4743 6.2893 68.0567 88.6572 curve; high efficacy10 85 Complete −5.5 2.8473 1 102.022 0 1.1 0 0 0 0 101.819 0 11.396494.1328 101.819 curve; high efficacy 11 85 Complete −5.4 2.5334 0.9997101.384 −0.509 1.1 0 0 0 0 101.791 0 7.5981 83.8704 101.791 curve; highefficacy 12 85 Complete −5.55 1.4781 0.9999 102.305 9.3996 1.1 0 0 0 0100.299 12.3078 36.4535 84.576 100.299 curve; high efficacy 13 84Complete −5.35 3.9295 0.9991 94.7101 −27.2857 1.1 0 0 0 0 95.6495−24.7665 −27.3506 80.6874 95.6495 curve; high efficacy 14 84 Complete−5.65 2.3332 0.9995 62.8391 −21.9345 1.1 0 0 0 0 61.7295 −21.6121 3.541659.0823 61.7295 curve; high efficacy 15 84 Complete −5.5 1.6259 0.999975.2612 −16.1195 1.1 0 0 0 0 73.6236 −13.6594 4.7051 58.0277 73.6236curve; high efficacy 16 84 Complete −5.65 1.3443 0.9999 77.492 −37.85681.1 0 0 0 0 75.2018 −30.2295 5.6414 58.1985 75.2018 curve; high efficacy17 84 Complete −5.6 1.2475 0.9998 71.4307 −24.7641 1.1 0 0 0 0 67.6004−18.3073 9.3675 52.6808 67.6004 curve; high efficacy 18 84 Partial −54.9549 0.9995 1760.36 8.7984 2.1 0 0 0 0 1767.43 7.745 30.584 357.8451767.43 curve; high efficacy 19 84 Complete −5.85 1.3437 0.9992 52.7174−28.8963 1.1 0 0 0 0 52.0677 −20.1458 14.9627 43.8719 52.0677 curve;high efficacy 20 84 Complete −5.45 2.3031 0.9999 83.8587 −17.7204 1.1 00 0 0 82.8998 −18.1348 −4.4619 69.3278 82.8998 curve; high efficacy 2184 Complete −5.25 4.4495 0.9992 93.4556 1.1731 1.1 0 0 0 0 93.2691 2.8560 75.5731 93.2691 curve; high efficacy 22 84 Complete −5.35 4.95490.9999 83.1353 −0.2899 1.1 0 0 0 0 82.8991 0 0 78.2915 82.8991 curve;high efficacy 23 84 Complete −5.4 2.9023 0.9998 98.4745 13.3122 1.1 0 00 0 98.87 12.6255 18.8414 87.3421 98.87 curve; high efficacy 24 84Complete −5.5 2.1211 0.9996 82.7045 5.7274 1.1 0 0 0 0 81.3384 5.886519.6198 73.0848 81.3384 curve; high efficacy 25 84 Complete −5.65 1.66040.9995 64.1141 −18.7854 1.1 0 0 0 0 63.0097 −16.5079 11.1354 54.533663.0097 curve; high efficacy 26 84 Complete −5.7 1.1 1 59.5556 −36.18071.1 0 0 0 0 56.0664 −25.4416 4.5885 41.9493 56.0664 curve; high efficacy27 83 Complete −5.7 2.2526 0.9994 54.9511 −26.8995 1.1 0 0 0 0 53.7617−26.1631 3.3138 51.6127 53.7617 curve; high efficacy 28 83 Complete−5.55 2.5334 0.9999 55.3257 −36.6914 1.1 0 0 0 0 54.6586 −35.9928−20.5758 48.8507 54.6586 curve; high efficacy 29 83 Complete −5.4 1.78850.9997 55.7271 −25.4387 1.1 0 0 0 0 54.9055 −23.8063 −13.4709 35.959154.9055 curve; high efficacy 30 83 Complete −5.4 2.1876 0.9999 73.6408−19.157 1.1 0 0 0 0 73.4371 −18.3179 −9.0684 55.4601 73.4371 curve; highefficacy 31 83 Complete −5.6 1.9673 0.9999 62.1733 −24.2621 1.1 0 0 0 062.3312 −22.9164 −0.943 53.2493 62.3312 curve; high efficacy 32 83Complete −5.55 2.1211 1 60.2337 −28.5623 1.1 0 0 0 0 59.833 −28.0565−9.4589 51.0458 59.833 curve; high efficacy 33 82 Complete −5.7 1.10.9991 33.1603 −50.1655 1.1 0 0 0 0 31.0963 −41.8046 −14.3237 16.969131.0963 curve; high efficacy 34 65 Complete −5.85 1.8851 0.9995 76.35525.9015 1.2 0 0 0 0 75.3947 9.4093 43.8029 74.3361 75.3947 curve; partialefficacy 35 64 Complete −5.6 3.132 0.9995 72.5763 −0.1751 1.2 0 0 0 071.7515 0 12.0467 71.5303 71.7515 curve; partial efficacy 36 64 Complete−5.5 1.4163 0.9997 81.2047 5.5 1.2 0 0 0 0 78.5607 7.6747 25.712965.0516 78.5607 curve; partial efficacy 37 64 Complete −5.6 2.40640.9991 74.4523 −2.5935 1.2 0 0 0 0 72.867 −1.7745 15.279 70.7278 72.867curve; partial efficacy 38 63 Complete −5.35 4.9549 0.9992 73.5449−2.0156 1.2 0 0 0 0 72.6726 −0.6248 −2.8833 69.1705 72.6726 curve;partial efficacy 39 63 Complete −5.45 1.7529 0.9998 69.5378 −9.0026 1.20 0 0 0 69.0926 −7.7641 5.5069 52.9348 69.0926 curve; partial efficacy40 48 Partial −4.85 4.5045 0.9986 433.479 −3.212 2.1 0 0 0 0 427.494−13.7066 5.577 25.2218 427.494 curve; high efficacy 41 48 Partial −4.854.5045 1 456.749 8.8874 2.1 0 0 0 0 453.124 8.7547 9.7092 34.1487453.124 curve; high efficacy 42 48 Partial −4.85 4.9549 0.9987 484.8839.6207 2.1 0 0 0 0 481.034 20.1308 0 32.0577 481.034 curve; highefficacy 43 47 Partial −5.4 0.9 0.9999 148.01 0 2.1 0 0 0 0 131.21413.6807 43.9438 94.2627 131.214 curve; high efficacy 44 47 Partial −4.954.095 0.9997 310.669 −17.6073 2.1 0 0 0 0 308.204 −13.4703 −18.759838.1168 308.204 curve; high efficacy 45 47 Partial −4.85 4.5045 0.9989419.509 −7.0263 2.1 0 0 0 0 413.716 −16.6135 0.0514 18.6133 413.716curve; high efficacy 46 46 Partial −5.1 1.01 0.9992 195.813 −35.6022 2.10 0 0 0 156.151 −29.6685 4.4407 77.5979 156.151 curve; high efficacy 4746 Partial −5.25 1.331 0.9993 153.844 12.5272 2.1 0 0 0 0 142.44817.1767 32.1719 97.3497 142.448 curve; high efficacy 48 46 Partial −5.151.7137 0.9992 178.694 −35.4511 2.1 0 0 0 0 165.458 −32.0109 −23.292376.5902 165.458 curve; high efficacy 49 46 Partial −5.25 1.9282 0.9996153.971 2.973 2.1 0 0 0 0 150.952 2.3042 15.4629 102.178 150.952 curve;high efficacy 50 46 Partial −5.1 1.8851 0.9989 186.381 5.0579 2.1 0 0 00 176.497 7.9011 9.4014 94.1426 176.497 curve; high efficacy 51 46Partial −5 4.9549 0.9988 249.6 −23.8252 2.1 0 0 0 0 249.102 −27.9345−17.6035 31.4284 249.102 curve; high efficacy 52 46 Partial −5.35 1.47810.9998 137.586 −27.7073 2.1 0 0 0 0 130.29 −25.6891 1.731 86.3564 130.29curve; high efficacy 53 45 Partial −5.15 0.8 0.9991 152.283 −11.267 2.10 0 0 0 118.6 0 28.0603 71.9463 118.6 curve; high efficacy 54 45 Partial−4.9 1.1341 1 262.045 39.3368 2.1 0 0 0 0 213.045 42.0153 58.6176119.863 213.045 curve; high efficacy 55 45 Partial −4.9 4.9549 0.9985252.825 −18.4203 2.1 0 0 0 0 252.32 −24.5803 −12.1219 3.459 252.32curve; high efficacy 56 45 Partial −5.4 1.5095 0.9999 110.65 21.8862 2.10 0 0 0 107.846 23.9844 38.0808 86.6615 107.846 curve; high efficacy 5745 Partial −5.35 1.7529 0.9999 115.173 −0.5596 2.1 0 0 0 0 112.9141.0034 14.5781 82.1622 112.914 curve; high efficacy 58 45 Partial −5.450.9 0.999 95.6282 −42.3624 2.1 0 0 0 0 82.8269 −29.6769 3.3459 49.086982.8269 curve; high efficacy 59 45 Partial −5 3.5722 0.9991 185.351−3.6299 2.1 0 0 0 0 183.88 0 −6.1928 48.4893 183.88 curve; high efficacy60 45 Partial −5.35 1.7529 0.9992 116.389 −12.5146 2.1 0 0 0 0 115.466−10.9556 4.4512 78.2781 115.466 curve; high efficacy 61 45 Partial −5.31.1705 0.9991 136.382 −3.94 2.1 0 0 0 0 126.28 −0.1233 25.8337 82.3962126.28 curve; high efficacy 62 45 Partial −5.15 1.9673 0.9992 162.393−23.864 2.1 0 0 0 0 154.66 −21.3534 −18.0137 77.9021 154.66 curve; highefficacy 63 45 Partial −5 3.1925 0.9993 198.688 −1.7425 2.1 0 0 0 0195.944 −3.9508 2.2361 57.8488 195.944 curve; high efficacy 64 45Partial −5.3 2.4729 0.9996 129.946 −4.9734 2.1 0 0 0 0 128.914 −6.39973.462 95.0937 128.914 curve; high efficacy 65 45 Partial −4.7 4.0950.9991 501.215 9.2299 2.1 0 0 0 0 464.087 8.214 2.5941 27.1667 464.087curve; high efficacy 66 45 Partial −4.9 4.5045 0.9991 227.168 11.91752.1 0 0 0 0 225.365 16.1816 8.6821 32.7652 225.365 curve; high efficacy67 44 Partial −5 1.4163 0.9999 162.351 4.7344 2.1 0 0 0 0 141.667 6.39814.4975 69.8045 141.667 curve; high efficacy 68 44 Partial −4.9 4.50450.9986 221.625 −17.9914 2.1 0 0 0 0 219.866 −11.823 −22.0487 4.4563219.866 curve; high efficacy 69 44 Partial −5.4 1.3987 0.9999 88.0721−2.436 2.1 0 0 0 0 84.3602 0 16.1931 62.7677 84.3602 curve; highefficacy 70 44 Partial −5.35 1.2876 0.9999 96.1281 −19.1057 2.1 0 0 0 089.5048 −15.4839 4.1098 57.1687 89.5048 curve; high efficacy 71 44Partial −5.1 1.3443 0.9999 122.331 −2.1806 2.1 0 0 0 0 109.03 0 9.629658.7582 109.03 curve; high efficacy 72 44 Partial −5.05 2.3332 0.9987136.354 −2.3089 2.1 0 0 0 0 132.126 0.9098 −2.4849 54.5396 132.126curve; high efficacy 73 44 Partial −4.9 4.095 0.9993 186.64 16.3113 2.10 0 0 0 185.158 13.4604 18.6219 35.6621 185.158 curve; high efficacy 7444 Partial −5.35 1.8617 1 86.169 −23.105 2.1 0 0 0 0 84.2199 −22.8223−9.7273 56.677 84.2199 curve; high efficacy 75 44 Partial −5.4 1.55790.9999 98.3982 26.9551 2.2 0 0 0 0 95.9047 28.0842 39.9836 79.709395.9047 curve; partial efficacy 76 44 Partial −5.2 1.3443 0.9997 105.907−8.518 2.1 0 0 0 0 96.9841 −5.8134 5.6525 55.1076 96.9841 curve; highefficacy 77 44 Partial −5.35 1.8617 1 87.1172 −34.2328 2.1 0 0 0 085.0608 −33.466 −19.7149 54.711 85.0608 curve; high efficacy 78 44Partial −5 1.7137 0.9999 162.598 9.6566 2.1 0 0 0 0 148.085 10.134115.4808 69.8478 148.085 curve; high efficacy 79 44 Partial −5.35 1.34370.9999 84.8433 −0.3646 2.1 0 0 0 0 80.1706 1.5242 16.235 57.3461 80.1706curve; high efficacy 80 44 Partial −5 1.4163 0.9999 169.032 −6.484 2.1 00 0 0 145.969 −4.7753 4.5639 65.9307 145.969 curve; high efficacy 81 44Partial −4.9 4.5045 0.9998 216.419 3.1931 2.1 0 0 0 0 214.702 1.82725.1171 24.2861 214.702 curve; high efficacy 82 44 Partial −5.5 1.1341 189.9991 −6.4745 2.1 0 0 0 0 84.7449 0 23.1094 63.7317 84.7449 curve;high efficacy 83 44 Partial −4.95 2.0937 1 162.065 −12.9886 2.1 0 0 0 0149.231 −13.5366 −10.5766 41.5268 149.231 curve; high efficacy 84 44Partial −5.2 1.2221 0.9999 114.278 −7.9639 2.1 0 0 0 0 102.4 −5.290511.0012 59.7194 102.4 curve; high efficacy 85 44 Partial −5.3 1.86170.9999 102.938 3.2331 2.1 0 0 0 0 100.92 3.2508 13.2564 71.6469 100.92curve; high efficacy 86 44 Partial −5.35 0.8 0.9999 94.6981 0.4963 2.1 00 0 0 80.4147 10.3775 28.9147 57.3965 80.4147 curve; high efficacy 87 44Partial −5.3 2.1211 0.9997 87.756 21.3502 2.2 0 0 0 0 86.5444 20.876126.4772 69.1414 86.5444 curve; partial efficacy 88 43 Partial −5.05 1.010.9999 98.4045 9.418 2.1 0 0 0 0 81.7691 12.5888 21.7607 50.5291 81.7691curve; high efficacy 89 43 Partial −5.3 1.6924 0.999 75.3022 −10.89572.2 0 0 0 0 74.1053 −9.9886 −0.2967 45.5813 74.1053 curve; partialefficacy 90 43 Partial −4.95 4.5045 0.999 146.447 5.7331 2.1 0 0 0 0146.154 2.8256 8.1166 27.152 146.154 curve; high efficacy 91 43 Partial−4.95 4.095 0.9986 122.391 −16.7058 2.1 0 0 0 0 121.42 −19.1946 −13.33746.5712 121.42 curve; high efficacy 92 43 Partial −5.35 1.6259 0.999976.9712 1.7602 2.2 0 0 0 0 74.9657 2.5679 13.3008 54.89 74.9657 curve;partial efficacy 93 43 Partial −4.95 1.7529 1 114.284 −3.6365 2.1 0 0 00 101.857 −3.4693 0 36.266 101.857 curve; high efficacy 94 43 Partial −54.9549 0.9996 138.031 9.6823 2.1 0 0 0 0 137.756 11.4997 8.6578 36.5266137.756 curve; high efficacy 95 43 Partial −5.15 1.01 0.9993 83.3763−14.8966 2.2 0 0 0 0 67.941 −12.4138 3.0575 36.2186 67.941 curve;partial efficacy 96 43 Partial −5.35 1.9887 0.9998 74.9858 −8.0822 2.2 00 0 0 73.1974 −7.8942 1.1034 54.4732 73.1974 curve; partial efficacy 9743 Partial −5.25 3.132 0.9994 79.8262 1.2108 2.2 0 0 0 0 79.2643 1.90741.3725 59.0705 79.2643 curve; partial efficacy 98 43 Partial −5 4.5045 1125.633 −0.0479 2.1 0 0 0 0 125.383 −0.0399 0.1051 28.903 125.383 curve;high efficacy 99 43 Partial −5 2.1211 0.9999 121.073 0 2.1 0 0 0 0114.004 0.7442 1.7316 44.4339 114.004 curve; high efficacy 100 43Partial −5.4 1.01 0.9992 64.0202 −31.5116 2.1 0 0 0 0 56.3462 −24.5207−4.9865 30.1418 56.3462 curve; high efficacy 101 43 Partial −5.05 1.66040.9989 115.659 −15.8708 2.1 0 0 0 0 104.197 −14.2124 −11.5581 43.2955104.197 curve; high efficacy 102 43 Partial −4.9 4.5045 0.9999 160.17110.4042 2.1 0 0 0 0 158.9 10.2684 11.2981 24.3058 158.9 curve; highefficacy 103 43 Partial −5.2 1.8265 0.9986 96.8922 −23.0045 2.1 0 0 0 091.3206 −22.0036 −16.3773 50.2037 91.3206 curve; high efficacy 104 43Partial −5 3.6772 0.9988 128.477 −9.1182 2.1 0 0 0 0 127.457 −6.4868−11.6559 27.3282 127.457 curve; high efficacy 105 43 Partial −5 3.51170.9995 113.218 16.8467 2.1 0 0 0 0 112.32 15.3847 17.8982 44.2423 112.32curve; high efficacy 106 43 Partial −5.15 1.21 1 79.2242 −2.155 2.2 0 00 0 70.115 −0.495 9.0228 40.4026 70.115 curve; partial efficacy 107 43Partial −5.35 1.4781 0.9998 76.3701 −19.2206 2.1 0 0 0 0 73.3496−17.2197 −3.2434 45.9779 73.3496 curve; high efficacy 108 43 Partial−4.9 4.5045 0.9999 142.05 2.0834 2.1 0 0 0 0 140.923 2.0308 3.142215.3462 140.923 curve; high efficacy 109 43 Partial −4.9 4.9549 0.9994174.079 7.5839 2.1 0 0 0 0 173.731 5.1999 10.0589 20.86 173.731 curve;high efficacy 110 43 Partial −4.9 4.5045 0.9999 162.565 −18.2174 2.1 0 00 0 161.275 −18.9356 −17.8813 0 161.275 curve; high efficacy 111 43Partial −5.15 1.21 0.9999 101.431 2.2101 2.1 0 0 0 0 89.9207 4.122415.4494 54.5855 89.9207 curve; high efficacy 112 43 Partial −5.25 3.1321 74.0416 0.1229 2.2 0 0 0 0 73.7713 0 1.5319 54.0265 73.7713 curve;partial efficacy 113 43 Partial −5 3.9295 0.9994 105.867 −5.8907 2.1 0 00 0 105.027 −4.9426 −7.7278 23.4771 105.027 curve; high efficacy 114 43Partial −4.95 4.5045 0.9989 134.828 −3.8223 2.1 0 0 0 0 134.559 −6.7171−1.2468 17.1799 134.559 curve; high efficacy 115 43 Partial −5.15 1.82650.9997 80.6607 −4.8829 2.2 0 0 0 0 77.6085 −3.9401 0 40.2449 77.6085curve; partial efficacy 116 43 Partial −5.05 1.5579 0.9999 94.7519−9.8246 2.1 0 0 0 0 84.9032 −9.1033 −3.9425 36.7288 84.9032 curve; highefficacy 117 43 Partial −5.1 1.6604 0.9998 88.7778 −0.2202 2.1 0 0 0 082.2017 0 5.0693 43.6203 82.2017 curve; high efficacy 118 43 Partial−5.3 1.6924 0.9992 74.8733 −7.9176 2.2 0 0 0 0 73.5555 −8.1247 2.29448.7391 73.5555 curve; partial efficacy 119 43 Partial −4.95 4.95490.9997 111.883 −16.0213 2.1 0 0 0 0 111.66 −17.1068 −14.6664 0.6285111.66 curve; high efficacy 120 43 Partial −5 0.8 0.9996 126.76 11.9332.1 0 0 0 0 97.3577 17.804 33.8372 63.3531 97.3577 curve; high efficacy121 43 Partial −5.05 1.8851 0.9997 97.7753 5.7133 2.1 0 0 0 0 92.59026.8559 8.4589 45.0425 92.5902 curve; high efficacy 122 43 Partial −51.2475 0.9999 127.656 0.5248 2.1 0 0 0 0 107.455 2.4969 11.4724 54.0343107.455 curve; high efficacy 123 43 Partial −5.05 1.3443 0.9999 96.90163.9836 2.1 0 0 0 0 85.4512 5.1227 12.1485 45.1238 85.4512 curve; highefficacy 124 43 Partial −5.05 1.2475 0.9999 102.977 −20.4393 2.1 0 0 0 085.8569 −19.2219 −7.3929 35.4594 85.8569 curve; high efficacy 125 43Partial −4.95 1.1705 0.9999 133.669 −2.6906 2.1 0 0 0 0 107.624 0 9.255850.3725 107.624 curve; high efficacy 126 43 Partial −5.05 1 1 125.1120.673 2.1 0 0 0 0 101.717 5.1764 18.9708 58.0571 101.717 curve; highefficacy 127 43 Partial −4.9 4.5045 0.9995 152.332 −18.9102 2.1 0 0 0 0151.123 −20.0375 −16.1406 −2.9507 151.123 curve; high efficacy 128 43Partial −4.85 4.5045 0.9991 153.542 14.6735 2.1 0 0 0 0 152.324 17.34112.5761 22.6146 152.324 curve; high efficacy 129 43 Partial −4.95 4.0950.9985 128.069 −17.8675 2.1 0 0 0 0 127.052 −21.7078 −15.591 8.9584127.052 curve; high efficacy 130 43 Partial −4.95 4.5045 0.9998 127.627.8122 2.1 0 0 0 0 127.365 7.1779 9.1804 26.1033 127.365 curve; highefficacy 131 43 Partial −4.85 4.9549 0.9997 183.761 8.7591 2.1 0 0 0 0182.302 10.6925 7.1202 16.169 182.302 curve; high efficacy 132 43Partial −4.9 4.9549 0.9996 133.42 12.8108 2.1 0 0 0 0 133.154 11.146113.8268 23.024 133.154 curve; high efficacy 133 42 Partial −5 2.72020.9992 73.1459 −27.5656 2.1 0 0 0 0 70.2432 −25.994 −28.4428 5.98570.2432 curve; high efficacy 134 42 Partial −5 4.4495 0.9993 72.9186−22.5632 2.1 0 0 0 0 72.8618 −20.9617 −23.9361 −0.0686 72.8618 curve;high efficacy 135 42 Partial −5 4.9549 0.9997 61.8199 −29.3022 2.1 0 0 00 62.005 −28.5367 −29.3616 −11.1775 62.005 curve; high efficacy 136 42Partial −5.05 0.8 0.9997 85.8101 −3.3079 2.2 0 0 0 0 65.1019 2.023114.5105 38.0502 65.1019 curve; partial efficacy 137 42 Partial −5 4.95490.9999 61.7369 −19.2861 2.1 0 0 0 0 61.7759 −19.2435 −19.3578 −1.669961.7759 curve; high efficacy 138 42 Partial −4.95 1.3437 1 107.998−27.3652 2.1 0 0 0 0 85.8466 −26.0685 −19.0518 23.5861 85.8466 curve;high efficacy 139 42 Partial −4.95 4.5045 0.9999 82.0264 14.712 2.1 0 00 0 81.696 15.171 14.5765 24.988 81.696 curve; high efficacy 140 42Partial −5 2.7868 0.9991 77.8665 −4.0391 2.2 0 0 0 0 75.9628 −2.7644−4.956 22.8114 75.9628 curve; partial efficacy 141 42 Partial −5 4.95490.9998 82.7722 −17.8968 2.1 0 0 0 0 82.663 −17.1706 −18.2787 2.603382.663 curve; high efficacy 142 42 Partial −5.05 2.4064 0.9988 71.1773−18.7775 2.1 0 0 0 0 68.9039 −17.1048 −18.6659 16.7527 68.9039 curve;high efficacy 143 42 Partial −5 4.4495 0.9999 81.2596 4.6719 2.1 0 0 0 080.844 4.2822 4.8849 22.4204 80.844 curve; high efficacy 144 42 Partial−4.9 4.095 0.9995 103.816 9.7776 2.1 0 0 0 0 102.992 8.5445 11.110220.4218 102.992 curve; high efficacy 145 42 Partial −5 4.9549 0.999970.4611 0.4668 2.2 0 0 0 0 70.5123 0.3023 0.1807 15.6647 70.5123 curve;partial efficacy 146 42 Partial −5 4.9549 0.9999 75.4854 −20.924 2.1 0 00 0 75.4881 −20.8468 −20.9197 0 75.4881 curve; high efficacy 147 42Partial −4.95 2.0937 0.9999 91.2034 1.0531 2.1 0 0 0 0 84.9194 0.45022.7027 29.0697 84.9194 curve; high efficacy 148 42 Partial −4.95 2.35310.9997 109.969 −28.7719 2.1 0 0 0 0 102.444 −30.0492 −26.8413 12.5122102.444 curve; high efficacy 149 42 Partial −5.05 1.111 0.9999 82.4023−1.9678 2.2 0 0 0 0 68.0415 −0.1259 8.5917 37.0328 68.0415 curve;partial efficacy 150 42 Partial −5 4.5045 0.9999 67.4005 −0.2112 2.2 0 00 0 67.1116 0 0.1599 15.1419 67.1116 curve; partial efficacy 151 42Partial −5 3.132 0.9999 65.1432 −26.3814 2.1 0 0 0 0 63.6617 −26.5586−25.9538 2.045 63.6617 curve; high efficacy 152 42 Partial −4.9 1.13410.9999 88.7605 −8.4801 2.2 0 0 0 0 67.3709 −6.7254 0 26.2892 67.3709curve; partial efficacy 153 42 Partial −5.1 1.5095 0.9998 74.4536−21.2671 2.1 0 0 0 0 66.0451 −20.7719 −14.2846 26.1958 66.0451 curve;high efficacy 154 42 Partial −5.5 0.9 0.9999 47.5155 −46.353 2.1 0 0 0 038.9061 −36.1249 −14.4105 17.8641 38.9061 curve; high efficacy 155 42Partial −5 3.99 0.9998 60.936 −32.1225 2.1 0 0 0 0 60.5343 −32.0766−31.7653 −9.1234 60.5343 curve; high efficacy 156 42 Partial −5 2.53340.9999 67.8023 −0.0336 2.2 0 0 0 0 65.3359 0 0 22.9027 65.3359 curve;partial efficacy 157 42 Partial −5 3.6272 0.9989 68.2216 −1.9559 2.2 0 00 0 67.4685 −0.7059 −3.266 17.7416 67.4685 curve; partial efficacy 15842 Partial −5 1.9282 0.9994 70.7327 −0.5234 2.2 0 0 0 0 65.8674 0.67050.6338 26.3415 65.8674 curve; partial efficacy 159 42 Partial −4.954.095 0.9995 106.672 −2.9033 2.1 0 0 0 0 105.826 −4.2541 −1.9112 17.1057105.826 curve; high efficacy 160 42 Partial −4.95 4.095 0.9998 100.584−2.4804 2.1 0 0 0 0 99.7859 −2.4977 −3.3563 16.0042 99.7859 curve; highefficacy 161 42 Partial −5.4 1.1 0.9998 55.0498 −42.9418 2.1 0 0 0 048.073 −37.0265 −17.738 22.4249 48.073 curve; high efficacy 162 42Partial −5.25 2.2526 1 55.2524 −31.8121 2.1 0 0 0 0 54.2827 −31.8337−27.5288 26.3243 54.2827 curve; high efficacy 163 42 Partial −5 4.0450.9999 78.2931 −0.8551 2.2 0 0 0 0 78.1035 −0.8037 −0.3894 19.426178.1035 curve; partial efficacy 164 42 Partial −5 4.5045 0.9998 91.8250.8371 2.1 0 0 0 0 91.6418 1.1551 0.0123 22.043 91.6418 curve; highefficacy 165 42 Partial −4.95 4.5045 0.9999 108.575 −20.12 2.1 0 0 0 0107.713 −20.5064 −20.4325 0 107.713 curve; high efficacy 166 42 Partial−4.9 1.3987 0.9998 104.163 10.3691 2.1 0 0 0 0 87.6791 10.2974 15.557841.6567 87.6791 curve; high efficacy 167 42 Partial −5.15 1.7885 0.999548.5966 −38.2233 2.1 0 0 0 0 43.999 −38.1941 −33.4689 9.5564 43.999curve; high efficacy 168 42 Partial −5.05 1.9673 0.9989 82.861 −0.78052.2 0 0 0 0 77.8049 0 35.7996 77.8049 curve; partial efficacy 169 42Partial −5 4.4495 0.9987 64.8483 −9.7104 2.1 0 0 0 0 64.3252 −18.1947−21.5953 0.301 64.3252 curve; high efficacy 170 42 Partial −5 4.44950.9988 87.8075 3.4732 2.1 0 0 0 0 87.6323 2.3641 5.3324 22.1704 87.6323curve; high efficacy 171 42 Partial −5 3.132 0.9999 69.5258 9.9159 2.2 00 0 0 68.362 10.0449 10.2067 28.1342 68.362 curve; partial efficacy 17242 Partial −5 2.3332 0.9987 73.8043 8 2.2 0 0 0 0 71.1503 8.9457 7.223931.1775 71.1503 curve; partial efficacy 173 42 Partial −4.95 0.9 0.998793.0488 7.5952 2.2 0 0 0 0 72.2674 10.3627 21.0562 42.1173 72.2674curve; partial efficacy 174 42 Partial −4.95 1.8579 0.9993 83.6585−20.386 2.1 0 0 0 0 74.1998 −21.7408 −16.656 13.5733 74.1998 curve; highefficacy 175 42 Partial −5.25 1.3443 0.9985 54.0029 −62.687 2.1 0 0 0 048.1163 −59.8895 −45.4644 5.3909 48.1163 curve; high efficacy 176 42Partial −5 4.9549 0.9985 96.131 −19.4361 2.1 0 0 0 0 95.8578 −22.0943−16.8813 5.2636 95.8578 curve; high efficacy 177 42 Partial −5 4.95490.9998 71.5284 0.9153 2.2 0 0 0 0 71.1427 1.3136 0.2953 16.2008 71.1427curve; partial efficacy 178 42 Partial −4.95 1.1341 0.9994 85.3274−22.6542 2.1 0 0 0 0 64.2561 −21.1531 −11.2645 18.8833 64.2561 curve;high efficacy 179 42 Partial −5.25 2.2526 0.9998 48.386 −35.6913 2.1 0 00 0 46.8236 −35.3915 −32.1742 20.8832 46.8236 curve; high efficacy 18042 Partial −5 3.1925 0.9991 76.0607 −1.6238 2.2 0 0 0 0 75.0675 0−2.3977 21.2822 75.0675 curve; partial efficacy 181 42 Partial −4.94.9549 1 93.0239 12.5 2.1 0 0 0 0 92.8383 12.3946 12.9169 18.884792.8383 curve; high efficacy 182 42 Partial −5 2.2481 0.9998 98.2495−0.8571 2.1 0 0 0 0 93.5709 0 0 34.7191 93.5709 curve; high efficacy 18342 Partial −5 4.4495 0.9997 99.728 1.0471 2.1 0 0 0 0 99.5289 0 2.034624.4291 99.5289 curve; high efficacy 184 42 Partial −5 2.9023 0.999885.5304 −0.6011 2.1 0 0 0 0 83.8533 0 −0.5506 26.0285 83.8533 curve;high efficacy 185 42 Partial −5 4.9549 0.9994 94.2492 −23.4705 2.1 0 0 00 94.548 −21.6443 −24.0045 0 94.548 curve; high efficacy 186 42 Partial−5.4 0.9 0.9999 47.9827 −45.5302 2.1 0 0 0 0 37.2481 −37.158 −17.6915.0968 37.2481 curve; high efficacy 187 42 Partial −5.05 1.3437 0.999265.7373 −54.3669 2.1 0 0 0 0 51.5472 −53.6324 −42.397 −1.7858 51.5472curve; high efficacy 188 42 Partial −5 4.5045 0.9999 88.2262 0.195 2.1 00 0 0 88.0501 0 0.7946 19.9555 88.0501 curve; high efficacy 189 42Partial −5.05 0.8 0.9999 84.5737 8.5392 2.2 0 0 0 0 66.7349 13.140523.7073 43.9763 66.7349 curve; partial efficacy 190 42 Partial −4.952.0937 0.9991 87.524 −28.9068 2.1 0 0 0 0 79.582 −30.5111 −25.26266.3098 79.582 curve; high efficacy 191 42 Partial −4.95 4.9549 0.999896.2795 −19.3188 2.1 0 0 0 0 96.0338 −18.0752 −19.9657 −4.197 96.0338curve; high efficacy 192 42 Partial −4.95 4.095 0.9992 76.4906 2.47892.2 0 0 0 0 76.1007 0.8898 2.9636 15.9079 76.1007 curve; partialefficacy 193 42 Partial −4.95 4.095 0.9997 92.2616 2.547 2.1 0 0 0 091.5294 3.7365 1.9996 17.8759 91.5294 curve; high efficacy 194 42Partial −5.05 1.7137 0.9999 73.9084 −2.9581 2.2 0 0 0 0 68.0475 −2.42570 30.6498 68.0475 curve; partial efficacy 195 42 Partial −5 4.045 0.998789.5777 −9.8792 2.1 0 0 0 0 89.3989 −7.8811 −12.0365 15.2586 89.3989curve; high efficacy 196 42 Partial −5 2.8473 0.9999 66.5821 −0.5091 2.20 0 0 0 65.0912 0 0 20.6484 65.0912 curve; partial efficacy 197 42Partial −5.05 0.9 0.9999 84.7038 1.5 2.2 0 0 0 0 66.8554 5.2461 15.703140.796 66.8554 curve; partial efficacy 198 42 Partial −5 4.9549 0.999379.1775 1.0914 2.2 0 0 0 0 79.0861 2.3908 0 17.574 79.0861 curve;partial efficacy 199 42 Partial −4.9 1.2876 0.9988 96.2012 0.1862 2.2 00 0 0 78.1385 0 7.6087 32.1891 78.1385 curve; partial efficacy 200 42Partial −5.05 1.4781 0.9999 56.5654 −42.0662 2.1 0 0 0 0 46.2423−40.8885 −35.7652 1.5723 46.2423 curve; high efficacy 201 42 Partial−4.95 2.3531 0.9987 74.9126 −19.8816 2.1 0 0 0 0 69.7901 −21.5136−17.1084 8.0866 69.7901 curve; high efficacy 202 42 Partial −5 1.86170.9999 80.5565 −3.3861 2.2 0 0 0 0 74.538 −3.4095 −0.6635 28.063 74.538curve; partial efficacy 203 42 Partial −5.15 1.4781 0.9998 59.3901−47.2995 2.1 0 0 0 0 51.7897 −45.9905 −36.9465 8.4225 51.7897 curve;high efficacy 204 42 Partial −5 3.5722 0.9999 70.7642 0.4864 2.2 0 0 0 070.2936 0.8815 0.1572 20.0962 70.2936 curve; partial efficacy 205 42Partial −5.15 1.4781 0.9999 57.7575 −40.2978 2.1 0 0 0 0 50.1627−39.3964 −31.6886 12.0722 50.1627 curve; high efficacy 206 42 Partial−4.95 4.095 0.9985 91.8921 13.9528 2.1 0 0 0 0 91.1628 12.1077 15.271828.5203 91.1628 curve; high efficacy 207 42 Partial −5 2.5334 0.999875.4708 0.1699 2.2 0 0 0 0 73.3009 0.545 0 25.4562 73.3009 curve;partial efficacy 208 42 Partial −5 3.1925 0.9988 74.5741 −9.1413 2.2 0 00 0 73.4157 −7.8355 −10.7764 16.4066 73.4157 curve; partial efficacy 20942 Partial −4.8 4.095 0.9997 142.41 12.1779 2.1 0 0 0 0 138.801 12.960110.613 18.5675 138.801 curve; high efficacy 210 42 Partial −4.9 4.95490.9986 113.27 11.9156 2.1 0 0 0 0 113.044 13.8203 9.4299 20.2893 113.044curve; high efficacy 211 42 Partial −4.95 1.1341 0.9991 109.674 −0.2652.1 0 0 0 0 87.88 0.2296 11.6146 43.0472 87.88 curve; high efficacy 21242 Partial −4.85 4.5045 0.9993 128.55 9.0119 2.1 0 0 0 0 127.53 11.12757.371 15.9241 127.53 curve; high efficacy 213 42 Partial −4.95 1.28760.9999 109.228 −1.0122 2.1 0 0 0 0 90.1221 0 6.4924 41.084 90.1221curve; high efficacy 214 42 Partial −4.95 2.0937 0.9997 108.196 13.90342.1 0 0 0 0 101.307 13.239 16.0649 43.8718 101.307 curve; high efficacy215 42 Partial −5 2.1211 0.9999 87.4293 13.9023 2.1 0 0 0 0 83.259714.2367 15.1789 40.8331 83.2597 curve; high efficacy 216 42 Partial −50.8 0.9996 94.4089 3.67 2.2 0 0 0 0 71.6749 8.2756 20.8299 43.990571.6749 curve; partial efficacy 217 42 Partial −5 3.0654 0.9995 66.18139.7958 2.2 0 0 0 0 65.2014 10.407 9.2853 27.4661 65.2014 curve; partialefficacy 218 42 Partial −5 3.6272 0.9998 71.8402 13.7566 2.2 0 0 0 071.4891 13.9594 13.1115 29.7899 71.4891 curve; partial efficacy 219 42Partial −4.95 1.5936 0.9993 76.3682 −7.5793 2.2 0 0 0 0 66.0085 −8.2231−3.0469 21.4195 66.0085 curve; partial efficacy 220 42 Partial −4.953.2975 0.9995 95.08 8.1688 2.1 0 0 0 0 93.7673 7.1434 9.5135 27.635693.7673 curve; high efficacy 221 41 Partial −4.95 4.095 0.9994 66.5046 72.2 0 0 0 0 66.1162 6.3785 7.8673 16.8498 66.1162 curve; partialefficacy 222 41 Partial −4.9 4.095 0.9999 66.8444 9.8519 2.2 0 0 0 066.4815 9.4439 9.7686 16.5469 66.4815 curve; partial efficacy

Example 3

In this example, embodiments of small molecules that increase α7integrin in skeletal muscle are disclosed. Using myogenic cells frommice in which the LacZ reporter gene was inserted into exon 1 of themouse α7 integrin gene, 403,000 compounds were screened and more than1500 hits that increased the β-galactosidase reporter were identified.Further evaluation identified 6 compounds that increased α7 integrin atleast 1.5-fold in myotubes. Compounds were classified as iron chelatingcompounds, cell cycle inhibitors and compounds with undefined function.Compounds identified from this screen represent novel molecular probesthat can be used to further elucidate regulation of α7β1 integrinexpression and signaling in skeletal muscle and may serve as potentialtherapeutics for the treatment of DMD.

Several therapeutic approaches have been developed with the aim ofrestoring dystrophin expression and shown efficacy in animal models ofDMD. These include virally mediated delivery and expression ofdystrophin, myoblast cell transfer and engraftment, exon-skipping andstop-codon read-through. Currently, none of these methods have beenapproved as therapy for DMD patients. An alternative approach is totarget and enhance levels of proteins which modify disease progressionand act to partially compensate for the absence of dystrophin. Thesedisease modifiers include utrophin, IGF-1, α7β1 integrin, GalNac, nNosand Adam12.

The α7β1 integrin is a laminin receptor in skeletal muscle that servesto link laminin-211/221 in the basal lamina and the actin cytoskeletonof muscle. The α7β1 integrin has structural and signaling functions thatcontribute to muscle development and physiology and was originallyidentified as a marker for muscle differentiation. Studies have shownthat enhanced transgenic expression of the α7 integrin in skeletal andcardiac muscle can ameliorate dystrophic pathology and extend thelifespan of mdx/utr^(−/−) mice more than three-fold. Multiple mechanismsappear to contribute to α7 integrin mediated rescue of dystrophindeficient muscle including maintenance of myotendinous and neuromuscularjunctions, enhanced muscle hypertrophy and regeneration, and decreasedapoptosis and cardiomyopathy Enhanced α7 integrin also protects musclesagainst exercise-induced damage. Conversely loss of the α7 integrin inmdx mice results in more severe muscle disease. Together theseobservations support the idea that the α7β1 integrin is a major diseasemodifier in DMD.

To translate transgenic mouse studies into potential therapies for DMD,a drug discovery program to identify chemical probes that increase α7integrin in skeletal muscle was initiated. A report on the generationand characterization of an α7 integrin knockout line of mice in whichthe LacZ gene is inserted into intron 1, downstream of the endogenous α7integrin promoter has been reported. Thus, β-galactosidase functions asa reporter for α7 integrin expression in these animals. Primary myogeniccells were isolated from a heterozygous mouse (α7βgal^(+/−)) so that thecells express α7 integrin and also report for transcription of theintegrin. The myogenic reporter cells were designated α7βgal^(+/−) andwere used to identify two molecules, valproic acid and laminin-111, inpreliminary screens and have been successfully tested in mouse models ofDMD.

To identify further small molecules that increase α7 integrin inskeletal muscle, a muscle cell-based assay to screen 403,000 compoundsincluding FDA approved drugs and the large compound libraries at theNational Chemical and Genomics Center (NCGC) was used. Several compoundsthat increase the α7 integrin with known mechanisms of action includingiron chelators, microtubule inhibitors, cell cycle inhibitors andsteroid-like molecules were identified. Additionally, several smallmolecules with unknown biological activities were identified. Overall,the results identified novel small molecules that increase the α7integrin in cultured muscle cells and may serve as molecular probes tofurther dissect signaling pathways that regulate the α7β1 integrin inskeletal muscle. These small molecules could potentially be developed asnovel therapeutics in the treatment of Duchenne and other fatal musculardystrophies.

Cell Culture:

C2C12 myoblasts and myotubes were grown as previously described.α7^(+/LacZ) myoblasts were originally isolated and maintained asdescribed. Briefly, myoblasts were grown and maintained in DMEM withoutphenol red (Sigma) containing 20% FBS (Atlanta Biologicals), 1%Penicillin/Streptomycin (P/S) (GIBCO)+L-Glutamine (GIBCO). Myoblastswere maintained below 70% confluence until use in assay. Myoblasts weredifferentiated into myotubes in DMEM without phenol red, 1% horse-serum(Atalanta Biologicals), and 1% P/S+L-Glutamine. All cells were incubatedat 37° C. with 5% CO₂. Assays were performed on myoblasts and myotubesbetween passages 8 and 14.

Compound Libraries:

Four compound libraries were screened using our muscle cell basedassay: 1) Prestwick Chemical and Microsource Spectrum Libraries(BioFocus DPI, Leiden Netherlands with facilities in UK, Basel,Heidelberg) (Overington et al., 2006). 2) DIVERSet library (ChembridgeCorp., San Diego, Calif.) and compounds from the ChemDiv library. 3)LOPAC library (Sigma-RPI) consists of 1280 pharmaceutically activecompounds. 4) MLSMR—Molecular Libraries Small Molecule Repository.

Myoblast α7^(+/LacZ) Integrin FDG Assay:

A total of 5000 α7βgal^(+/−) myoblasts were dispensed in 100 μL growthmedia using a 12-well multi-pipette (Rainin) onto Nunc black sided TCcoated 96-well plate. After 24 hours up to 1 μl of compound in DMSO wasadded to the myoblast plates from pre-made working drug plates using a 1μl 96-well pin tool or using an 8-well automatic multichannel pipette.Each working drug plate contained a column of a positive control (sodiumbutyrate (Fluka) or SU9516 (Tocris)) and at least one column containingDMSO alone. After incubating for 48 hours the media was aspirated, andcells were lysed with 504 of Mammalian Protein Extraction Reagent (MPER)(Thermo) per well followed by incubation at room temperature for 10minutes. β-galactosidase (βgal) activity in each well was quantified byadding 50 μL of FDG assay solution (20% 0.1M sodium phosphate buffer pH7.0 (Sigma), 0.2% 1M MgCl₂ (Sigma), 0.2% 20 mM fluoresceindi-galactoside (FDG) (Marker Gene Technologies)) and incubating theplates in the dark for 20 minutes at room temperature. Stop solution(2×TE) was then added (100 μl/well) and plates were read forfluorescence on the Victor V (Perkin-Elmer) with an excitation filter at485 nm, an emission filter at 535 nm, and a 0.1 s/well count time.

Myotube α7 integrin FDG assay: A total of 25,000 α7βgal^(+/−) myoblastswere dispensed in 1004 growth media. After 24 hours, growth media wasaspirated, wells were washed with 2004 PBS, and 100 μL/well ofdifferentiation media was added. Differentiation media was changed dailybetween 72 and 120 hours, and up to 1 μL compounds in DMSO were added aspreviously described once wells contained differentiated myotubes. TheFDG fluorescence assay was performed as described in the myoblast screenwith the one notable exception being the incubation after FDG solutionaddition being shortened from 20 minute to 5 minutes at RT due to thehigher levels of βgal in myotubes.

qHTS of the LOPAC Library and MLSMR:

A total of 250 α7LacZ^(+/−) myoblasts at passage 13 were dispensed usinga multidrop (Thermo) into black low base tissue culture treatedmicroclear aurora plates in 6 μl media containing DMEM without phenol,5% FBS, 1× GluMax and 1× Penicillin/Streptomycin. The plates wereincubated 16-24 hours at 37 C, 5% CO2, 95% humidity covered with lowevaporation stainless steel lids from Kalypsys. Compounds were thendispensed using a Kalypsys pintool to deliver 23 nl/well compounds inDMSO (diluted into 6 μl resulting in a 1:260 dilution of compound). Thepositive control compound used was the cdk2 inhibitor SU9516 (Tocris)(identified in the initial LOPAC screen). The plates were incubated for48 hours at 37 C, 5% CO2, 95% humidity using the same stainless steellids. After incubation with compound, 5 μl of the media was aspiratedusing the Kalypsys washer/dispenser and 3 μl of the Mammalian ProteinExtraction Reagent (MPER) lysis buffer (Thermo Fisher) was added. Theplates were spun at 2000 rpm to remove bubbles and an initial capturewas acquired on the Viewlux (Perkin Elmer) with excitation at 480 nm andemission at 540 nm for 25 seconds to omit any auto fluorescentcompounds. The plates were then incubated for 10 minutes at roomtemperature. After incubation, 3 μl of 125 μM FDG (Marker GeneTechnologies) diluted in PBS with 2 mM MgCl₂ and 0.2% β-mercaptoethanol(BME) was added, the plates were then centrifuged at 2000 rpms andincubated for 30 minutes at room temperature. The plates were again readon the Viewlux with excitation at 480 nm and emission at 540 nm for 25seconds.

CMV-LacZ Secondary Screen:

In order to determine compounds that acted to stabilize β-gal inmyogenic cells, a CMV-LacZ C2C12 cell line was used in a secondaryscreen. The EGFP gene from the pEGFP (Clontech) vector was replaced byLACZ PCR product with digests of the vector/PCR product performed usingEcoRI (NEB) and NotI (NEB) and ligated using T4 DNA Ligase (NEB)following standard procedures to produce the CMV-LACZ vector. The LACZgene was generated by PCR from the pBK-RSV (Stratagene) vector usingprimers LACZ EcoRI F- and LACZ NotI R-. The CMV-LACZ vector was thendigested with AfIII (NEB) to linearize the construct, which was thentransfected in C2C12 myoblasts using Lipofectamine 2000 (Invitrogen)following the manufacturer's instructions. Stable cell lines wereselected in growth medium containing G418 (Life Technologies) at 1.5mg/ml. Clonal lines were selected and screened for βgal levels using themyoblast FDG assay. C2C12 cell lines with the highest βgal expressionwere used to screen cherry picked compounds, following the sameprocedures defined above for the myoblast and myotube screens. Compoundswere determined to have failed this secondary assay if signal waselevated above the average DMSO+2× standard deviation (˜30%) for twoseparate data points.

Western Blotting:

C2C12 myotubes were treated for 48 hours with each hit compound, washedwith PBS, then scraped and pelleted using standard procedures. Cellpellets were resuspended in RIPA buffer and protein concentrationdetermined by BCA. Approximately 10 μg of protein was loaded per laneand separated on 8% SDS-PAGE gels and transferred to nitrocelluloseusing standard conditions. Blots were probed using α7A and α7B integrinspecific rabbit polyclonal antibody as previously described andnormalized to α-tubulin.

Statistical Analysis and Curve-Fitting:

Statistical analysis was performed using Graphpad Prism software andunpaired t-test comparison against the DMSO control treatment group.Graphpad prism software was also used to fit curves using nonlinearregression analysis with log (agonist) vs. response with a variableslope. A constraint equal to 1 was placed on the bottom of the curve andeither 2 or 2.5 at the top (when needed) in order to produce appropriateEC₅₀ values.

Generation and validation of α7^(+/LacZ) integrin myoblast assay: Inorder identify α7 Integrin enhancing compounds, an assay based onα7^(+/LacZ) mouse derived primary myoblasts and myotubes was developed.Heterozygous myoblasts were used in order to maintain a copy of theItga7 gene for normal myogenic cell adhesion and signaling. The secondItga7 gene allele had exon 1 replaced by the LacZ gene, providing areporter for α7 integrin transcription and maintaining the endogenouspromoter, enhancer, and chromatin environment. By deriving primarymuscle cells, myogenic cells capable of differentiating into myotubesand in which the β-gal reporter levels accurately mimicked the α7integrin protein levels during this developmental process were produced.Several rounds of pre-plating were performed with the myogenic line usedfor the screen in order to remove fibroblasts, leaving a relatively purepopulation of myogenic cells.

α7 Integrin Muscle Cell-Based Assay:

Assessing the growth rate of myogenic cells in a 96-well format wascarried out. For the myoblast assay, it was determined that plating 5000α7^(+/LacZ) cells led to a confluence of ˜80% at the time offluorescence quantification. Limiting the confluence to less than 80%helped prevent entry into a differentiated state in this assay which mayaffect LacZ expression. For the myotube assay, it was determined thatplating 25,000 myoblasts led to a confluence of around 95% after 24hours allowing myogenic differentiation to proceed. Next, the linearityof fluorescence for the assay was determined for various amounts ofpurified E. coli β-galactosidase using the FDG assay solution (FIG. 4A).This displayed a linear response that was comparable to levels observedin the α7^(+/LacZ) myoblasts. Finally, as library compounds are normallysolubilized in DMSO, the effects of DMSO on our assay were examined forboth myoblasts and myotubes (FIG. 4B). In myoblasts, DMSO concentrationsof up to 0.5% had no significant effect on the assay compared to cellswithout DMSO addition (FIG. 4C). At 1% DMSO, there was a 10% decrease inoverall fluorescent signal in the assay, which was considered to bewithin acceptable range (FIG. 4C). The effect of DMSO on the myotubeassay was more robust, where 0.5% DMSO decreased fluorescence by ˜12%and 1% DMSO displayed ˜25% decrease in overall fluorescence (FIG. 4B).Greater than 1% DMSO resulted in reduced fluorescence and therefore 1%DMSO in media was determined to be the maximum acceptable level formyotubes. The levels of FDG were optimized to 125 μM which was used inthe MLSMR screen and subsequent verification assays (FIG. 4D).

For our preliminary screens, sodium butyrate was determined to increaseβ-galactosidase levels with treatments of ˜1.5 mM. In initial studieswith sodium butyrate a Z′ factor of 0.6, which is considered a strongassay for drug discovery, was calculated and thus a myotube screen ofthe Prestwick Chemical at 0.2 ug/mL and Microsource Spectrum Librariesat 10 μM was initiated. As these libraries are relatively small,myotubes were initially screened because they are the ideal therapeutictarget for α7 Integrin elevation. Using a 1.2 fold increase over DMSOcontrol as the minimum cutoff, 24 compounds in the Prestwick chemicallibrary and 30 compounds in the Microsource spectrum library wereidentified. These corresponded to a hit percentage of 2.1% and 1.5% ofthe libraries. After secondary and counter screens, the compounds thatincreased α7 integrin were classified as either iron chelators or acholesterol analog. The iron chelators identified in the screen wereCiclopirox ethanolamine, deferoxamine, and 2,2-Dipyridyl which alldisplayed positive dose-response curves in myotubes. The cholesterolanalog compound, 5alpha-cholestan-3β-ol-6-one, also displayed a positivedose-response curve.

This initial success led us to attempt a larger scale high-throughputscreen (HTS) using the DIVERSet library of compounds with sodiumbutyrate as a positive control. Due to the number of compounds in theDIVERSet library (˜50,000), a myoblast screen was performed. This screengenerated several more “hits” which upon secondary screens were narroweddown to three compounds annotated: 1001, 1002, and 1003. After furtherstudies, these compounds and analogs were either less robust or tootoxic to move forward with further studies.

Primary Screen of LOPAC Library and the MLSMR:

The assay was adapted for a high-throughput screen with the fullyautomated Kalypsys robotic system using 1536 well plates at NCGC. Apreliminary screen of the LOPAC library with 1001 (DIVERSet) as apositive control was used to develop the new conditions for this assay,comparing offline to online results and reducing myoblasts plated to2000 cells/well for the 384-well format. This screen produced several“hit” compounds including Sodium Nitroprusside dihydrate, a Nitric oxidedonor, and two cdk2 inhibitors SU9516 and CK2 inhibitor 2. SU9516 gave arelatively robust response of around 2.1-fold, relative to DMSO alone.There was still a relatively high variability in DMSO background signal(˜30%) even using SU9516 as a positive control. However, as SU9516 wasthe most reproducibly active drug that had been identified it wasselected as a positive control for the qHTS. To this end a dose-responsecurve SU9516 treatment of myoblasts and myotubes was generated (FIGS. 5Aand 5B).

Using SU9516 as a positive plate control and DMSO as a negative control,the assay was then used to screen the Molecular Libraries Small MoleculeRepository (MLSMR) of 368,680 compounds at NIH Chemical Genomics Center(NCGC) using concentrations at 0.08 μM, 0.4 μM, 2 μM, and 10 μM. Around1500 compounds were cherry picked as “hits” based on previously definedcurve classifications (FIG. 6).

Offline Confirmation and β-Galactosidase Stability Secondary Screen:

From the 1500 cherry picked “hits” two pools were chosen for furtheroff-line myoblast, myotube and secondary screening. The first poolconsisted of 166 compounds, which were still active in a subsequentonline myoblast screening of the original 1500 cherry picks (Tables9-12). The second pool consisted of the top 197 compounds, which had notbeen repeatedly active in subsequent screens (Tables 9-12). Finally, asSU9516 had proven to be a highly reliable positive control, 44 analogs,based on the SU9516 platform, were selected from the MLSMR library orfrom separate stock compounds and screened. The secondary screenconsisted of a CMV-LACZ stably transfected C2C12 myoblast line withconsistent β-Galactosidase activity (Tables 9-16, and Table 17). Theassays were used the original 96-well format and new dose-responsecurves for myoblasts (N=3-6 replicates for each data point), myotubes(N=3-6 replicates for data point), and CMV-LacZ secondary assay (N=2replicates for each data point) using concentrations of 0.5 μM, 1 μM, 5μM, 10 μM, 20 μM, and 40 μM (Tables 9-16). A cutoff of greater than 25%relative to DMSO was used to exclude compounds (Table 17). Almost onefifth of the compounds from the initial 197 top “hits” workedexclusively in myotubes assay and not in myoblasts. Together, this datasuggested that during the primary screen some of the cells in the assayhad differentiated into myotubes. This trend was not observed in the 166NCGC compounds where almost one third worked exclusively in myoblasts.Again, during the transition from on-line to off-line screening many ofthe compounds from both groups (54% and 69%, Table 17) failed to achievethe minimum cutoff of 25% increase over DMSO levels in either myoblastsor myotubes. In addition the majority of compounds, which showedactivity in the β-galactosidase stabilizing secondary screen, did notshow activity in either of the α7^(+/LacZ) based assays (Table 17, fullresults in Tables 9-12). These results suggest some of these compoundsmay not be actually stabilizing or catalyzing the β-galactosidaseenzymatic reaction but instead may be activating the CMV promoter in theC2C12 cells.

As SU9516 was used as our positive control, it was used to potentiallyrepresent a platform that could be modified to produce other “hit”compounds. off-line screens with 44 SU9516 analogs were performed. Themyoblast, myotube, and secondary CMV-LacZ assays were performed aspreviously described and results from this screen are summarized inTable 17 (Full results in Tables 13-16). Surprisingly, many of them, aswith SU9516 itself, showed activity in the C2C12 CMV-LACZ myoblasts andmyotubes. Most of the compounds had activity in the β-galactosidasestabilizing screen also had no response in the primary screen suggestingthat they were not β-galactosidase stabilizers but instead activators ofCMV or the region of CMV-LACZ stable integration within this cell line.This result also suggests that SU9516 was likely activating both theITGA7 promoter and the CMV-promoter by inhibition or activation ofseparate signaling pathways. With reference to Tables 9-16 below, valuesof less than about 0.9 correspond to less desirable compounds ascompounds with such values were toxic at the indicated concentrationvalue; values of 0.9 to about 1.1 indicate compounds with some activity(substantially similar to the DMSO background); and values greater thanabout 1.2 indicate compounds that have desirable activity as thesevalues indicate compounds exhibiting an activity about 20% above thebackground.

TABLE 9 Burkin Assay in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000591667-01 1.1351 0.86235 1.088 1.13163 1.03356 0.99007MLS000568234-01 1.01115 1.10558 1.37321 1.13643 1.03188 1.09422MLS000689562-01 0.88167 0.95366 1.22402 1.01263 1.014 1.07412MLS000732652-01 0.87916 1.0346 1.2648 0.97202 1.19119 1.17557MLS001240181-01 1.19808 1.15299 1.07353 1.4585 1.08072 1.08233MLS001211139-01 1.21551 1.218 0.96826 1.06491 1.13586 1.2392MLS001030268-01 0.60686 0.72598 0.87162 1.48967 1.14329 1.23909MLS000912699-01 0.43922 0.6724 0.9298 0.93216 1.03847 1.11521MLS001125260-01 0.90571 1.02411 0.96159 0.98381 1.11006 1.20955MLS000717689-01 0.98659 0.95999 1.03846 1.03203 1.04195 1.15829MLS001197665-01 1.18868 1.09447 0.95234 1.01544 1.06912 1.06224MLS001075922-01 0.64637 0.56776 0.2869 0.60386 1.29157 1.2085MLS001124046-01 1.07367 1.10106 1.01243 1.07966 1.15704 1.06118MLS001197220-01 1.0836 1.11129 0.92184 1.84318 1.28622 1.2811MLS001221318-01 1.07461 1.05387 0.99442 1.00961 1.28345 1.11823MLS000947910-01 1.01847 1.04651 1.0429 1.03277 1.18767 1.02381MLS001215795-01 0.71873 0.74097 0.77762 0.93196 1.20673 1.14257MLS002163670-01 0.7779 0.89724 0.94682 1.01239 1.23366 1.11688MLS001200149-01 0.98756 1.02136 0.99804 1.0782 1.15573 1.03945MLS001359861-01 0.91135 0.87253 0.93239 0.94838 0.93763 0.93526MLS000710669-01 0.82425 1.84768 0.99409 1.0722 1.04969 1.02525MLS001035690-01 1.09083 1.05474 0.98721 1.18564 1.15072 1.05147MLS001030621-01 1.00997 1.00129 1.16138 1.15044 1.15833 1.00359MLS001083082-01 0.94677 1.01412 1.02292 1.05814 0.95159 0.90356MLS000045588-01 0.89188 1.00313 0.91698 1.07484 1.10703 1.01584MLS001216939-01 0.78651 0.86335 0.98283 1.11114 1.2827 1.12283MLS001163859-01 0.92799 0.92082 1.02427 1.05488 1.09001 0.97855MLS000683232-01 1.65393 1.82061 1.77787 1.6445 1.19868 1.23351MLS001170856-01 0.71333 0.92168 1.24442 0.97435 0.86511 0.94996MLS002667707-01 0.83137 0.93474 1.23469 1.01997 0.91716 1.0156MLS001200665-01 1.02863 1.14332 1.24505 1.00302 0.8539 0.89824MLS002161853-01 0.9354 1.07259 1.1978 0.93857 0.82784 0.85687MLS002163101-01 0.97754 0.94568 1.25085 0.96995 0.94422 0.95515MLS000062431-01 0.9038 1.78859 1.20925 1.01602 0.95817 0.9542MLS000028160-01 0.85354 1.10681 0.85212 0.98053 0.98728 1.03731MLS002248819-01 0.76886 0.81835 1.0849 1.06786 1.11265 1.12574MLS000080654-01 0.91656 1.11208 0.94286 0.92349 0.92297 0.97875MLS000760876-01 0.95041 1.10218 0.91131 1.81188 0.98558 1.0287MLS000677675-01 0.95543 1.00416 1.02813 0.98734 1.06053 1.09282MLS000113985-01 0.12198 0.17793 0.48041 0.78982 1.01847 1.06463MLS001182368-01 0.89517 1.00108 0.89449 0.91403 1.21452 1.08093MLS001212882-01 1.04785 1.06823 0.9069 1.04855 1.06975 1.0132MLS001004364-01 0.93735 0.97287 0.95313 1.03485 1.10452 1.0917MLS000736846-01 0.95628 1.0406 1.01151 1.06976 1.2858 1.21826MLS001098105-01 1.13456 1.07472 0.97405 0.99364 1.02871 0.94487MLS000678673-01 1.13357 1.17252 0.96866 1.04367 1.05388 0.93249MLS000925023-01 0.41794 0.77654 0.79566 0.95232 1.03462 0.99596MLS001212319-01 0.78199 0.85293 0.88263 0.94115 1.02595 1.04963MLS000779126-01 0.93895 0.91244 0.81956 0.92292 1.11482 1.01879MLS000948055-01 0.58675 0.82128 0.8986 1.13571 1.05347 1.32788MLS000110418-01 0.8889 1.00365 1.11988 1.24866 1.07925 1.3095MLS000693704-01 0.80188 0.84732 0.94181 1.1427 1.15286 1.42821MLS001225512-01 1.28384 1.10796 0.88323 1.17276 0.91313 1.1596MLS001006798-01 1.01583 1.11843 1.00833 1.13293 0.92818 1.19095MLS000711491-01 0.57934 0.79492 1.02377 1.02034 1.0159 1.21405MLS000582947-01 0.77374 0.9417 0.98066 0.94304 0.96323 1.23612MLS000531177-01 0.80079 1.01406 1.62515 1.06657 0.92304 0.88259MLS001202389-01 0.25563 0.58161 1.1004 1.00911 0.94807 0.9323MLS000536064-01 0.45011 0.54402 1.04652 0.73332 0.80395 0.80279MLS000586245-01 1.02068 1.03337 1.24403 1.11633 1.04937 0.99033MLS001061374-01 0.67743 0.87297 1.16907 0.98264 0.84475 0.84766MLS000675441-01 0.89965 0.90155 1.24817 1.04359 0.8764 0.93971MLS001200396-01 0.4794 0.48269 1.1321 0.53784 0.92449 0.88271MLS001165937-01 0.68201 1.03268 1.70065 1.27472 0.89489 0.86889MLS000325736-01 0.88542 0.91197 1.16957 1.42277 0.78755 0.77572MLS001215357-01 0.53275 0.9203 1.48859 0.88374 0.93 1.03703MLS000588210-01 0.96107 1.13586 0.98164 0.90019 0.97069 1.05483MLS000764729-01 0.72603 0.9581 0.96935 0.98159 1.0582 1.08805MLS000689492-01 0.96521 1.08264 0.94309 0.93998 0.90795 1.07297MLS001000299-01 0.77383 0.93971 0.97497 0.95516 0.99529 1.08932MLS000393762-01 0.46958 0.59613 0.69566 0.77991 0.94283 0.97379MLS001130011-01 0.91554 1.03734 0.97619 1.37412 0.89472 0.97145MLS001229477-01 0.90194 0.96342 0.94542 1.36703 0.82957 0.87031MLS000707378-01 1.49252 1.31687 1.07579 1.02429 1.04559 1.00348MLS000573208-01 0.62351 1.63233 0.81207 0.95231 1.01493 1.03104MLS001167281-01 0.86182 0.88689 0.97761 1.07478 1.18016 1.08931MLS000053342-01 1.02205 1.0354 0.86681 0.97655 1.08832 1.22195MLS002171615-01 0.61782 0.70899 0.88587 0.97469 0.94068 1.05229MLS001005712-01 0.76395 0.87627 0.87827 0.92598 1.17003 1.1047MLS001176153-01 1.12011 1.14544 0.87665 0.91849 1.04463 1.06004MLS000735021-01 0.99412 1.02509 0.97651 0.99818 0.92462 0.94018MLS000767397-01 0.97544 1.61335 0.93339 1.47579 0.83903 0.88624MLS001196572-01 0.9345 0.93622 0.94826 1.22354 0.76263 0.88263MLS000393966-01 0.96498 0.99041 1.07573 1.79279 0.96263 0.94947MLS001034810-01 0.84068 0.8996 1.19071 1.37466 0.99747 1.02099MLS001165394-01 1.00709 1.03548 0.95105 1.15909 1.20071 1.14858MLS000089464-01 0.39111 0.56309 0.7825 0.93396 0.98019 0.99651MLS000698617-01 0.89657 0.84622 0.93172 1.06001 1.00295 1.00774MLS001175021-01 0.95749 0.97466 0.90603 0.88063 0.98034 0.92001MLS001166758-01 0.61737 0.72753 0.82445 0.97363 0.73944 0.82402MLS001008109-01 0.83059 0.82603 0.86263 1.00193 0.78003 0.84815MLS001181936-01 0.32845 0.50999 0.82189 0.85734 0.75618 0.84427MLS000560266-01 0.55892 0.83206 1.13014 1.02573 0.85564 0.95298MLS001215074-01 0.59739 0.73389 0.97384 0.94307 0.9004 1.02349MLS001215123-01 0.52524 0.665 1.15451 1.1241 0.97081 1.07545MLS001033255-01 0.82795 0.98503 1.12416 1.01684 0.93725 1.01302MLS001160611-01 0.92674 0.97495 1.11549 1.00379 0.97673 1.1058MLS001006302-01 0.94141 0.97305 1.17248 0.95352 0.93033 1.12331MLS001123876-01 0.88436 0.93393 1.09536 1.43977 0.82637 1.04105MLS001122698-01 0.83886 0.9061 0.96519 1.00842 0.85728 0.98289MLS000755214-01 0.86097 0.84475 0.96348 0.97565 1.02077 1.07667MLS000731285-01 0.60769 0.60402 0.7344 0.77267 0.92346 1.06384MLS000776409-01 0.34443 0.50867 0.98382 0.93888 1.00738 1.1194MLS001221908-01 0.56836 1.43328 0.88118 0.93061 0.95314 1.05089MLS000419286-01 0.71995 0.7945 0.8951 0.91739 1.00244 1.16649MLS000554416-01 0.82485 1.08704 0.9591 0.99152 1.0679 1.21133MLS000073150-01 1.06802 1.13888 0.90898 0.92622 1.0875 1.26179MLS000663185-01 0.83278 0.92216 0.93123 1.39854 0.97437 1.10863MLS001078811-01 0.74109 1.68071 0.89103 0.95834 0.99261 0.9376MLS002694363-01 0.70661 0.75811 1.08986 1.04452 1.06141 1.06241MLS000689218-01 1.03748 1.07006 1.05082 1.13165 1.17068 1.06835MLS001215294-01 0.20458 0.20253 0.61411 0.76405 1.00264 0.97828MLS001183575-01 0.53384 0.69163 0.79465 0.89805 1.00931 0.96443MLS000393567-01 0.87054 0.83339 0.93251 0.97236 1.05083 1.03233MLS000546316-01 1.02675 1.02594 1.12291 1.16412 1.22254 1.15942MLS000912258-01 0.37249 0.63513 0.84251 1.2722 0.90885 0.84356MLS000850522-01 0.88047 0.8743 1.00276 1.30679 0.93316 0.79932MLS001197779-01 0.75302 0.84665 0.98695 1.28053 1.0032 0.91613MLS001095705-01 0.72839 0.79174 0.969 1.29549 0.87876 0.78726MLS000912562-01 0.55515 0.69112 0.92532 1.03872 0.92904 0.76968MLS000092785-01 0.6509 0.78329 0.93433 1.1703 1.03537 0.95442MLS000418650-01 0.94454 0.91042 0.96326 1.12987 1.06885 0.91564MLS000860966-01 1.11657 1.24227 1.12552 1.22473 0.95061 0.8092MLS001180929-01 1.1941 1.10157 1.05028 1.04982 1.0555 1.07667MLS000564564-01 0.97011 0.94044 1.0834 1.04244 0.94502 0.99186MLS001164432-01 0.66217 0.7137 1.06663 1.09125 1.1813 1.24825MLS000621451-01 1.08838 1.0713 1.11408 1.06613 1.01829 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1.1396MLS001116535-01 1.26741 1.23894 0.93856 0.99796 1.11141 1.09197MLS001165424-01 1.22991 1.17612 0.95477 0.99659 1.2222 1.15184MLS001116079-01 1.34036 1.24425 0.9749 1.02479 1.08671 1.02952MLS001198271-01 1.0038 1.08798 1.03423 1.29177 1.00045 1.03412MLS001167798-01 1.00561 1.05069 1.06388 1.27705 1.16574 1.05836MLS000710288-01 0.59627 0.86808 1.1778 1.3928 1.30459 1.16807MLS000734270-01 0.98726 0.97392 0.95501 1.21022 1.04793 1.0135MLS000858981-01 1.00476 1.0113 1.01009 1.18572 0.999 0.95297MLS000698826-01 1.02313 0.99365 0.95981 1.24627 1.00831 1.01911MLS001000874-01 0.95449 1.00725 0.86772 1.07723 1.03283 1.17509MLS000682750-01 1.10263 1.12414 1.07554 1.22927 1.02005 1.20347MLS001090787-01 1.22203 1.26522 1.45989 1.26261 0.97343 1.14195MLS002636056-01 0.82025 0.93768 1.22389 1.03743 1.06589 1.15719MLS002170630-01 0.67353 0.78253 1.21616 1.0982 0.97082 1.12201MLS002162890-01 0.49245 0.694 1.26109 1.1338 1.03367 1.10651MLS001105912-01 0.46309 0.60252 1.24569 1.02884 1.05749 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0.96452MLS001033202-01 0.77044 0.84083 0.92815 0.99521 0.91528 0.66817MLS001216260-01 0.89335 0.87813 0.87981 0.8531 0.98403 1.0145MLS000085522-01 0.74815 0.97285 0.95306 1.11804 0.87205 0.99272MLS000702680-01 0.62643 0.69252 0.89075 0.86233 0.8804 0.84503MLS001212998-01 1.29981 1.36467 1.54392 1.45225 1.19984 1.19252MLS001160885-01 0.79137 1.02135 0.91473 0.94021 0.93557 1.05034MLS001122718-01 0.93319 0.99226 0.90919 1.03265 1.04066 1.1056MLS000027478-01 0.81604 0.91452 0.92087 0.97213 0.90438 0.95522MLS001177364-01 0.83857 1.00465 0.90148 0.96554 0.98693 1.08229MLS001179695-01 0.80683 0.89235 0.98712 0.87525 0.94414 1.03309MLS002251986-01 0.91837 0.97926 0.86667 0.95123 0.92314 1.04571MLS001166704-01 0.43935 0.57467 0.76522 0.84987 1.16515 1.10905MLS001196422-01 1.00409 1.24784 1.33031 1.22893 0.9784 0.74578MLS001179624-01 0.88278 0.87902 0.93143 0.95146 0.99629 0.99296MLS001223425-01 1.1318 1.22256 1.14626 1.05531 1.08325 1.05664MLS001117140-01 0.94202 0.9222 0.90096 0.90913 1.06544 1.02935MLS001110618-01 1.08304 0.97133 1.00452 0.9228 1.02989 0.95251MLS001223482-01 0.98024 1.28967 1.02296 1.0127 1.09469 1.06378MLS000680049-01 0.63536 0.74931 0.9727 0.96933 1.10296 0.99975MLS001212498-01 1.39711 1.28613 1.31206 1.18111 1.20238 1.05365MLS001124732-01 0.65809 0.68925 0.81246 0.8622 1.07116 1.12082MLS000526364-01 1.02864 1.11416 1.08517 1.04544 0.95015 0.97473MLS000767227-01 0.92827 0.91325 0.91067 0.85772 0.80731 1.02529MLS000703499-01 0.10433 0.44649 0.76322 0.9278 1.01689 0.70927MLS001167169-01 0.90615 0.81331 0.93783 0.95467 0.8858 1.03215MLS001198693-01 0.91638 0.93507 0.91367 0.92293 0.54265 0.96167MLS001219345-01 0.92275 0.98071 1.01406 1.04565 1.11947 0.30269MLS001211651-01 1.02624 0.99093 0.97202 1.01085 1.12405 1.06095MLS000806880-01 1.05558 0.99571 0.95237 1.00906 1.12862 1.09682MLS001223567-01 0.86396 0.85795 0.92033 0.96076 1.08244 1.04444MLS001005283-01 0.93009 0.89649 0.89081 0.93463 0.85822 0.87024MLS001218427-01 0.30387 0.22324 0.76631 0.83344 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1.11596MLS001080869-01 0.84846 0.97296 0.9857 1.14722 1.03572 1.14407MLS000332693-01 0.92254 0.89498 1.09531 1.06767 0.99365 1.03957MLS001141113-01 0.55984 0.91514 1.07109 1.07883 1.058 1.22948MLS001176611-01 0.91345 0.83982 1.07215 1.09667 0.99398 1.04935MLS001202627-01 0.21788 0.54268 0.77391 0.99233 1.08133 1.17187MLS000765108-01 1.02593 1.04146 1.11307 1.20183 1.1197 1.17146MLS000937079-01 0.99561 1.02838 1.14674 1.05254 0.89583 1.16289MLS001215742-01 0.81646 0.76822 1.10465 1.04044 0.92602 0.88704MLS001217045-01 1.10891 1.05719 1.11177 1.08675 0.96972 0.88104MLS001196946-01 0.08385 0.11943 0.61881 0.79887 0.97976 0.95406MLS001216714-01 0.27369 1.08646 1.13942 1.04321 0.97056 1.018MLS000772430-01 0.89631 0.88124 1.04602 1.00717 0.93622 0.97283MLS000693370-01 0.93667 0.95143 1.06427 0.88727 0.80004 0.86381MLS000769322-01 0.95504 1.00606 1.0771 0.96529 0.93537 0.95348MLS000721030-01 1.12244 1.02526 1.16581 1.02742 0.93485 0.9673MLS001176897-01 0.94746 0.94581 0.99783 0.90579 0.94565 0.9342MLS000774940-01 0.77609 0.85802 1.04275 1.09508 0.89283 0.93089MLS001030746-01 0.96367 0.93385 1.07333 1.06913 0.8373 0.91954MLS003126425-01 0.44013 1.12959 1.254 1.28169 1.1398 1.14604MLS001217697-01 1.0127 0.88306 1.15767 1.18252 1.11591 1.09067MLS000516719-01 0.96515 0.91967 1.06775 1.0321 1.04756 1.00574MLS001165323-01 0.47791 0.87431 0.92404 0.88215 0.94909 0.97044MLS001220803-01 0.99059 0.95026 0.95106 0.88777 1.0343 0.96878MLS001163121-01 0.91572 0.80739 0.99067 0.94885 1.01191 1.04852MLS001060561-01 0.63183 0.79502 0.88777 0.82662 0.92837 0.99611MLS001139515-01 0.85269 0.83359 1.15988 1.05338 0.98522 0.92246MLS001149811-01 0.61724 1.00026 1.02244 1.01027 0.88883 0.64967MLS000773700-01 0.87206 0.9227 0.86573 0.90585 1.0257 0.93354MLS001177045-01 0.95693 1.03087 1.0259 1.0025 1.03211 0.84633MLS000693747-01 0.91746 0.87957 1.08763 1.0596 1.03615 1.11428MLS001175556-01 0.80176 0.89561 0.88558 0.92019 0.86252 0.92004MLS001175473-01 0.86983 0.899 0.87154 0.87321 0.37213 0.53313MLS002156278-01 0.82456 0.86044 0.96531 1.00963 0.96419 0.85974MLS000707281-01 0.79693 0.83587 0.88542 0.89346 0.97563 0.95067MLS000591198-01 0.47304 0.77654 0.95931 1.03702 0.92511 1.00578MLS000714175-01 0.96263 0.99361 1.06 1.1625 0.97498 0.53831MLS002163386-01 0.93876 1.01374 0.88796 1.03415 0.9023 1.0508MLS000761297-01 0.95256 1.07174 1.05527 1.12127 1.07133 1.0693MLS002245351-01 0.86569 0.90007 0.97044 1.03385 1.03691 1.08244MLS000718886-01 0.81389 0.91243 0.96613 1.08727 0.93943 1.05198MLS002156485-01 1.02644 1.05206 0.87521 0.92779 0.9382 1.05917MLS001140657-01 0.79783 0.85676 0.87677 0.95283 0.96198 0.9918MLS002157024-01 1.08144 1.09694 0.85235 0.96094 0.97128 1.01144MLS000721730-01 0.93424 0.82698 0.88242 0.81574 0.81973 0.83765MLS000705922-01 0.87835 0.88922 0.72832 0.72819 1.02 0.96273MLS000724709-01 0.55365 0.48598 0.77902 0.73891 0.95538 0.98992MLS002161757-01 1.15126 1.00861 0.91351 0.91126 0.89196 0.96138MLS002164687-01 0.81941 0.83778 0.84902 0.69489 0.81935 0.77498MLS001060533-01 1.21969 1.39744 1.10126 1.03286 0.99152 0.90492MLS000685139-01 0.98483 0.98933 0.77595 0.84163 0.6285 0.82342MLS001217286-01 1.06759 1.00563 0.82726 0.76705 0.8506 0.74275MLS001221619-01 0.89756 0.96481 0.67637 0.73674 0.76333 0.68552MLS001219621-01 0.54233 0.78837 0.75418 0.72486 0.80633 0.86421MLS001166156-01 0.34807 0.75715 0.76012 0.75432 0.90852 0.8005MLS000534926-01 0.42215 0.59957 0.88325 0.85863 0.94733 1.52217MLS000548725-01 0.07694 0.51687 0.76446 0.89891 0.88938 0.91317MLS000374261-01 0.83211 0.83763 0.71381 0.72479 0.77583 0.7676MLS000123454-01 1.01341 1.51865 0.86209 0.78618 0.8234 0.8934MLS000625140-01 0.68287 0.75893 0.78335 0.87481 0.8709 0.88394MLS001214443-01 0.9313 0.9132 0.81732 0.7787 0.80738 0.92033

TABLE 10 Burkin Assay in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000591667-01 0.69646 0.67806 0.99863 1.07156 1.1434 1.00531MLS000568234-01 0.97051 0.86942 1.08971 0.96419 1.16742 1.07977MLS000689562-01 0.85465 0.72357 1.0159 0.97883 1.03466 1.08068MLS000732652-01 0.73092 0.67974 1.14125 1.1938 1.11599 1.09214MLS001240181-01 0.79047 0.88714 0.96827 1.64271 1.214 1.17431MLS001211139-01 0.75468 0.82834 0.94255 1.00287 1.28848 1.16726MLS001030268-01 0.76063 0.62773 0.92494 1.21276 1.16147 1.07576MLS000912699-01 0.65181 0.52169 0.93901 0.92519 0.93756 1.17498MLS001125260-01 0.74508 0.76341 1.09105 0.96768 1.20801 1.28483MLS000717689-01 0.71346 0.82829 1.07589 1.04848 0.90065 1.11085MLS001197665-01 0.95828 1.00065 0.99899 0.97302 1.06784 1.11547MLS001075922-01 0.82425 1.06745 0.62896 0.65563 1.14344 1.29247MLS001124046-01 0.91979 0.89276 1.01603 1.41708 1.09565 1.14517MLS001197220-01 0.83097 0.90004 1.00438 1.25409 1.3061 1.32659MLS001221318-01 0.7186 0.80714 0.95914 0.92662 1.08244 1.13276MLS000947910-01 0.95151 0.96285 1.03459 0.99998 1.10147 1.13447MLS001215795-01 0.88728 0.9147 0.91066 0.86305 1.19109 1.29108MLS002163670-01 0.73497 0.85264 1.07165 0.97782 1.29363 1.22297MLS001200149-01 0.83633 0.92859 1.09373 1.06886 1.07233 1.26026MLS001359861-01 0.80395 1.0337 0.876 0.90881 1.18375 1.09515MLS000710669-01 1.0073 0.99667 0.90001 1.05381 1.16308 1.04515MLS001035690-01 0.92376 0.97197 0.93577 1.07274 1.07159 1.11088MLS001030621-01 0.72029 0.7954 0.94292 0.99904 0.94925 0.97239MLS001083082-01 0.93703 0.89872 1.05034 1.07008 0.94106 1.03453MLS000045588-01 0.95463 1.06301 1.03566 1.135 1.09854 1.07688MLS001216939-01 0.47431 0.85294 1.06002 1.06991 1.18531 1.19276MLS001163859-01 1.05788 1.11822 1.24934 1.38468 1.28077 1.13173MLS000683232-01 1.31969 1.4149 1.44782 1.27158 1.2778 1.13572MLS001170856-01 1.02912 0.77626 1.06447 0.91001 0.74357 0.82959MLS002667707-01 0.7617 0.80494 0.95401 0.89392 1.01022 0.99551MLS001200665-01 0.88772 0.83653 1.12552 1.09065 0.84612 1.03137MLS002161853-01 0.93534 0.91896 1.15981 0.98887 0.80923 0.87612MLS002163101-01 0.97609 0.77823 1.30157 1.10149 0.90746 1.04549MLS000062431-01 0.88412 0.76433 1.1989 1.17804 0.84151 0.95709MLS000028160-01 0.81118 0.90493 0.95982 0.91617 1.10188 1.08464MLS002248819-01 0.85083 0.92428 1.24039 1.06554 1.19767 1.11701MLS000080654-01 0.62169 0.74219 0.97069 0.90438 1.09159 1.06711MLS000760876-01 0.89931 0.86336 0.9755 1.24882 1.25578 1.2427MLS000677675-01 0.68734 0.66754 1.04369 1.08103 1.20496 1.27844MLS000113985-01 0.66792 0.76552 0.99254 0.99189 1.03688 0.98825MLS001182368-01 0.91934 0.90974 1.02027 1.2117 1.0792 1.04351MLS001212882-01 0.91073 0.84206 0.8877 0.90721 1.02311 0.98143MLS001004364-01 1.05525 0.88751 0.86169 0.90474 1.10504 1.09276MLS000736846-01 0.96729 0.87764 0.93964 1.00375 1.14446 1.13093MLS001098105-01 0.84588 0.81422 0.83539 0.91598 1.05323 1.05204MLS000678673-01 0.78099 0.86554 0.96062 0.97673 0.98018 0.97725MLS000925023-01 0.73184 0.80043 0.8784 0.89694 1.23995 1.16045MLS001212319-01 0.63105 0.73964 0.92007 0.94764 1.13867 1.13422MLS000779126-01 0.77678 0.88319 0.87581 0.91457 1.01779 0.98357MLS000948055-01 0.75145 0.98002 0.8264 0.90529 1.05415 1.06716MLS000110418-01 0.88548 0.99394 0.943 1.01658 1.0975 1.07264MLS000693704-01 0.82627 0.9587 0.92299 1.00259 1.0787 0.93149MLS001225512-01 0.74015 0.84309 0.82346 0.97987 1.05854 0.92901MLS001006798-01 0.88856 0.97456 0.89057 1.05957 1.04218 0.93455MLS000711491-01 0.57898 0.67103 0.94615 0.9629 1.06241 1.06977MLS000582947-01 0.79101 1.10221 0.97595 1.01969 1.15528 0.90028MLS000531177-01 0.90051 0.80069 1.16345 1.05071 0.73291 0.82589MLS001202389-01 0.55936 0.64125 1.05346 0.99362 0.93925 0.9181MLS000536064-01 0.7073 0.76421 1.13143 0.95394 0.95554 0.90458MLS000586245-01 1.02629 0.94257 1.17916 1.04311 0.9584 1.00329MLS001061374-01 0.81546 0.73205 1.18149 1.00437 1.09204 1.05776MLS000675441-01 0.71678 0.74757 1.11902 0.95908 1.07053 0.85516MLS001200396-01 0.71138 0.6408 1.26379 0.98293 0.94875 0.93009MLS001165937-01 0.72485 1.00559 1.52391 1.21196 1.16352 0.96798MLS000325736-01 0.69718 0.6582 1.16824 1.02677 0.80167 0.91355MLS001215357-01 0.69286 0.75605 0.88397 0.90573 0.81367 0.9366MLS000588210-01 0.95653 0.97629 0.91553 0.94662 0.95586 0.94246MLS000764729-01 0.60751 0.68098 0.97198 1.00794 1.08392 1.05496MLS000689492-01 0.75436 0.88357 0.92917 0.93108 1.13846 1.15571MLS001000299-01 0.55782 0.793 0.91625 0.87644 1.00592 0.92249MLS000393762-01 0.70455 0.77047 1.00696 0.97947 0.92497 0.94021MLS001130011-01 0.7512 0.78831 1.0699 0.88339 0.99521 0.95531MLS001229477-01 0.8339 0.83764 0.90149 0.97022 0.93677 0.80147MLS000707378-01 1.05105 1.04623 1.24093 0.88992 0.85116 0.87159MLS000573208-01 0.59664 0.82393 1.04515 0.97999 1.00715 0.94525MLS001167281-01 0.83453 0.82926 1.14202 1.07436 1.05393 1.06349MLS000053342-01 1.11028 1.04947 1.03003 1.00173 1.01051 1.05896MLS002171615-01 0.83027 0.84318 0.99471 0.97089 1.16975 1.10717MLS001005712-01 0.74045 0.78848 0.93903 0.94721 1.17289 0.87044MLS001176153-01 0.98183 0.92376 1.10591 0.95378 1.02681 1.00708MLS000735021-01 0.85229 0.84443 1.00585 0.91367 1.00886 1.01265MLS000767397-01 1.01365 0.99933 1.0608 1.00428 0.94806 0.92939MLS001196572-01 0.71815 0.98051 0.92943 1.14827 0.88824 0.92363MLS000393966-01 1.01539 1.23304 0.95462 1.30422 0.84361 0.85472MLS001034810-01 0.75928 1.00003 1.16544 1.40257 1.06959 1.03516MLS001165394-01 1.03397 1.18823 0.98536 1.23914 1.13467 1.06778MLS000089464-01 0.83732 0.95449 0.99169 1.12764 1.02877 1.06322MLS000698617-01 0.79633 1.09199 0.94496 1.13858 1.07472 1.19659MLS001175021-01 0.87883 0.97988 0.99128 1.05123 1.0167 1.06135MLS001166758-01 0.87545 0.98563 0.89787 0.98565 1.02671 1.0327MLS001008109-01 0.61033 0.82635 0.93355 1.18189 0.91465 0.92469MLS001181936-01 0.73871 0.58684 1.02387 0.98561 0.76335 0.91972MLS000560266-01 0.60594 0.67057 1.18475 1.03662 0.88245 0.98908MLS001215074-01 0.84879 0.75046 1.13207 1.04996 0.83074 0.78865MLS001215123-01 0.816 0.69382 1.13745 1.04903 0.90099 0.94663MLS001033255-01 1.23171 0.83368 1.24617 1.08579 0.81878 0.90997MLS001160611-01 1.23985 0.83969 1.16888 1.00621 0.86282 0.94515MLS001006302-01 1.07828 0.78349 1.21228 1.03871 0.93734 0.98217MLS001123876-01 1.00321 0.71141 1.23169 1.1689 0.81926 0.86603MLS001122698-01 0.85722 0.92091 1.03016 0.95745 0.90389 0.91311MLS000755214-01 0.8312 0.96553 1.0353 0.93209 0.96607 1.00954MLS000731285-01 0.19528 0.68996 0.94092 0.86954 0.96101 0.85441MLS000776409-01 0.80563 0.89489 0.92835 0.88195 1.02936 0.89249MLS001221908-01 0.74062 0.81797 0.96928 0.88901 0.95887 1.19709MLS000419286-01 0.193 0.45703 0.94796 0.87898 0.8124 0.9511MLS000554416-01 0.49152 0.68156 0.93336 0.83662 0.9261 0.91456MLS000073150-01 0.85596 0.83687 0.94757 0.9467 0.92051 1.11147MLS000663185-01 1.11786 0.77913 1.0708 0.97669 0.81366 0.96799MLS001078811-01 0.45984 0.63069 0.8773 0.98157 0.90642 0.91635MLS002694363-01 0.41079 0.66943 0.87872 0.91782 1.01024 0.99095MLS000689218-01 0.96764 1.00798 0.98295 0.96996 1.08276 0.96628MLS001215294-01 0.8079 0.80622 0.82158 0.89346 1.10597 1.04694MLS001183575-01 0.68676 0.73778 0.79376 0.81721 0.96813 1.0887MLS000393567-01 0.92366 0.8607 0.88565 0.90647 0.99533 0.88817MLS000546316-01 1.17565 0.85707 1.09939 1.06571 1.17392 1.13749MLS000912258-01 0.71931 0.87695 0.96307 1.14382 0.85708 0.92635MLS000850522-01 0.93236 0.94157 0.92633 1.10981 0.90575 0.9439MLS001197779-01 0.91301 0.82374 0.92492 1.10225 0.96099 1.03803MLS001095705-01 0.79366 0.94108 0.8798 1.07342 0.94627 0.9916MLS000912562-01 0.70243 0.88725 0.8748 0.92895 0.85822 0.89803MLS000092785-01 0.82253 0.82933 0.85855 1.02029 0.96616 1.02722MLS000418650-01 0.75932 0.75808 0.96539 1.03182 1.00364 1.08596MLS000860966-01 0.89269 1.05975 1.10725 1.1137 0.82114 0.92753MLS001180929-01 1.13472 0.86586 1.02995 0.96426 0.92563 0.89896MLS000564564-01 1.08968 0.80854 1.05291 0.94262 0.94023 0.95865MLS001164432-01 0.84639 0.71209 0.99848 0.98784 0.95093 0.89821MLS000621451-01 1.02447 0.83274 1.07136 0.92535 0.8932 0.94711MLS000078709-01 0.48942 0.63113 0.98985 0.95354 0.82363 0.83742MLS000688267-01 1.13079 0.88079 1.10089 1.13158 0.83733 0.9011MLS001006516-01 0.71268 0.71266 0.90855 0.92575 0.87139 0.92612MLS001095033-01 0.91577 0.89377 0.92979 0.94401 0.94882 0.99586MLS000072290-01 0.17841 0.47499 0.84049 0.90961 1.12146 1.10865MLS000036988-01 0.10401 0.41718 0.82226 0.80994 0.75767 0.85532MLS001217935-01 0.52464 0.78867 1.08057 0.88116 1.01763 0.90754MLS000777780-01 0.77559 0.88145 0.93933 0.86968 0.98371 1.04663MLS001174740-01 0.70642 0.78226 0.90166 0.85739 0.86868 0.90778MLS000392555-01 0.93005 0.93498 0.97413 1.10516 0.96637 0.96465MLS000693795-01 0.95128 1.00214 0.93842 0.9381 0.91713 0.83969MLS000684034-01 0.73851 0.74026 0.80757 0.83826 0.95363 0.97308MLS001172822-01 0.78582 0.84656 0.86099 0.85134 0.93148 0.94964MLS000052969-01 0.55483 0.66209 0.876 0.91836 1.05823 0.91311MLS001217212-01 0.73655 0.76634 0.87723 0.88308 0.83859 0.82329MLS001004864-01 0.37599 0.60748 0.96185 0.97835 1.02518 0.949MLS001116535-01 0.83664 0.89931 0.85528 0.89286 1.00307 0.95003MLS001165424-01 0.76414 0.77905 0.83154 0.89381 0.85679 0.87714MLS001116079-01 0.96857 0.90267 0.90118 0.95586 0.95017 0.95237MLS001198271-01 0.82169 0.90557 0.98564 1.11296 0.9334 1.0083MLS001167798-01 0.72315 0.79831 0.93824 1.11791 0.92426 1.05071MLS000710288-01 0.8202 1.02449 0.95983 1.08387 0.95749 1.07633MLS000734270-01 0.81021 0.85578 0.90807 1.08322 0.89746 0.941MLS000858981-01 0.81733 0.85258 0.89224 1.05483 0.88365 1.02668MLS000698826-01 0.78436 0.95754 0.90009 1.08025 0.87178 1.00839MLS001000874-01 0.81807 0.92657 0.92162 1.0731 0.86464 0.9357MLS000682750-01 0.70644 1.00201 0.88388 1.07253 0.99385 0.99573MLS001090787-01 1.5544 1.20894 1.19518 1.08299 1.16762 1.05405MLS002636056-01 1.16657 0.90453 1.02703 0.92033 1.08895 0.96193MLS002170630-01 1.0714 0.88566 1.12282 0.96974 1.07532 1.03038MLS002162890-01 0.80295 0.7452 1.1501 0.93508 1.18455 1.05063MLS001105912-01 1.00319 0.82995 1.1322 0.94666 1.06085 0.94833MLS001007892-01 0.98876 0.83431 1.07453 0.97418 1.01306 0.90462MLS000089748-01 0.74149 0.77148 0.80258 0.8675 0.91388 1.04302MLS000912726-01 0.82375 0.82313 0.88602 0.88182 0.8889 1.06575MLS000086970-01 0.97145 0.96119 0.95762 0.86446 1.08763 1.00334MLS000420298-01 1.00617 0.91001 0.90053 0.94527 1.0464 0.95384MLS001147478-01 0.87441 0.8876 1.04143 0.99327 0.96771 1.07626MLS000090135-01 0.82149 0.8015 0.9078 0.87471 0.95976 1.04209MLS001179717-01 0.26634 0.5137 0.6812 0.74398 1.02658 0.8788MLS000683234-01 1.63139 1.41362 1.27328 1.09078 1.05264 0.9165MLS000695955-01 1.38633 1.23243 1.32243 1.14659 1.03159 0.94502MLS001125488-01 1.2197 1.14304 1.31932 1.11918 1.15129 1.03181MLS000768008-01 1.2867 1.09811 1.27255 1.10984 1.17937 1.07276MLS000913117-01 0.39105 0.49497 0.66235 0.96547 1.20782 1.08965MLS000860538-01 0.94028 0.96352 1.41241 1.31905 1.10205 0.98501MLS001177259-01 0.92374 0.81869 1.04363 0.99884 1.05301 0.94799MLS000861434-01 1.05784 0.87623 1.10144 0.97878 1.06594 0.95931MLS000047918-01 0.94474 0.96731 0.87188 0.8995 0.91346 1.40284MLS000389484-01 0.69458 0.86703 0.64333 0.8638 0.7793 0.92918MLS001217673-01 1.07849 1.09479 0.95155 0.96989 0.74898 0.88943MLS000389677-01 0.95301 0.64209 0.99562 1.10906 0.91563 1.00483MLS001208858-01 1.00672 1.09983 1.06192 1.10431 0.99771 1.09459MLS000333610-01 0.90959 0.87418 0.99324 1.00287 1.03921 0.98596MLS001117351-01 0.69855 0.76687 0.94632 0.83154 0.99239 1.02475MLS000682883-01 1.09375 1.10243 1.00996 0.99357 1.09503 0.81158MLS001095231-01 0.83555 0.98635 0.96948 1.02204 0.93816 1.00477MLS000721584-01 0.9927 0.91547 0.88669 0.88021 0.98327 0.91477MLS001183429-01 0.26288 0.4076 0.733 0.85574 0.82302 0.80419MLS002158881-01 1.00118 1.20183 0.77262 0.67034 0.86523 0.89603MLS001165749-01 0.73418 0.7808 1.12222 1.1624 1.07825 0.95317MLS001237320-01 0.97868 0.99122 0.65538 1.03546 1.05525 0.98786MLS000763405-01 1.37591 1.45481 1.1121 1.21073 1.13948 1.03273MLS000538580-01 0.18248 0.92803 0.47741 0.8641 1.02508 1.08678MLS001033202-01 0.98604 1.05145 1.0906 1.04232 1.01895 1.05691MLS001216260-01 0.93478 0.91445 1.06046 1.15971 0.98935 1.00499MLS000085522-01 0.556 1.02941 0.92486 0.87196 0.8415 0.96218MLS000702680-01 0.74641 0.80744 0.77734 0.96778 0.91962 1.01629MLS001212998-01 1.05241 1.4472 1.44543 1.4553 1.10305 1.14344MLS001160885-01 0.92003 1.01751 1.14627 1.10829 0.95686 1.00027MLS001122718-01 0.98039 1.08947 1.10635 1.06877 0.94347 1.01133MLS000027478-01 0.91147 0.98536 0.9583 0.984 0.90435 1.0222MLS001177364-01 0.6717 1.12523 1.21312 1.23479 0.95193 1.03584MLS001179695-01 0.85456 0.92985 0.99222 1.19881 0.94899 1.05344MLS002251986-01 0.91124 0.98819 1.08822 1.07325 0.95152 1.12062MLS001166704-01 0.81001 0.79177 1.06471 0.89357 1.26767 1.166MLS001196422-01 0.97494 1.09481 1.3198 1.19044 1.14189 0.97577MLS001179624-01 0.21827 0.89035 1.10439 1.04824 0.97089 0.9052MLS001223425-01 1.18449 1.14219 1.37641 0.91422 1.22038 1.07959MLS001117140-01 1.0628 1.08703 1.37473 1.09477 1.11707 0.93188MLS001110618-01 0.94634 1.0381 1.25185 1.11356 1.06727 0.95258MLS001223482-01 0.96702 1.13235 1.52904 1.26854 1.14804 1.06047MLS000680049-01 0.43791 0.87306 1.22022 1.04721 1.05615 0.88755MLS001212498-01 1.15666 1.15759 1.34193 1.10705 1.11751 0.94103MLS001124732-01 0.78881 0.87194 0.89636 0.87591 1.02949 1.0065MLS000526364-01 0.91621 1.35752 0.89076 1.03293 0.89526 0.96484MLS000767227-01 0.9434 0.77748 0.84403 0.96445 0.89975 0.85931MLS000703499-01 0.44935 1.10118 0.85178 1.05913 0.88093 0.97252MLS001167169-01 0.62616 1.3902 0.96963 1.04119 0.93745 0.89248MLS001198693-01 1.11598 0.93192 0.95144 1.01113 0.90482 0.95996MLS001219345-01 0.92318 0.71463 1.0424 1.07784 0.98891 0.94156MLS001211651-01 1.02681 1.07588 1.02524 1.11572 0.91297 0.85432MLS000806880-01 0.93527 1.05733 0.99601 0.90936 0.87308 0.735MLS001223567-01 0.99314 0.99913 0.92396 0.8636 0.99783 0.95334MLS001005283-01 0.75963 0.9432 0.91413 0.85832 0.99512 0.98114MLS001218427-01 0.42896 0.33115 0.60544 0.84638 0.7974 0.80515MLS001139288-01 0.78077 1.31295 0.97767 0.98285 0.85249 0.92343MLS000696445-01 0.84334 0.58427 0.62501 0.9714 0.86366 0.86019MLS001218795-01 0.96063 0.9463 0.99909 0.97775 0.9363 0.96478MLS000419555-01 1.01948 1.01177 1.09286 1.14975 1.01818 1.05246MLS001225507-01 0.95391 1.17494 1.00225 1.01286 0.67411 0.93211MLS000663651-01 0.96476 1.01671 1.00729 1.10579 0.90988 0.94596MLS000706349-01 0.92386 1.07347 0.9294 0.97383 1.00814 1.19252MLS000393110-01 0.81734 0.67258 0.98158 1.02232 0.92759 1.13888MLS000574647-01 0.82187 1.00598 0.85961 1.00656 0.86456 0.93501MLS000532969-01 1.46962 1.64768 1.35295 1.52016 1.03395 1.1906MLS001125260-01 0.90862 0.90566 0.94019 1.02222 0.86628 1.00234MLS000122749-01 0.93057 1.25564 1.13138 1.09637 0.99818 1.04233MLS001150751-01 0.5343 1.0842 1.02303 1.25375 0.95035 1.0993MLS001221867-01 0.9915 1.01963 0.93743 0.95928 0.94218 0.97278MLS001147727-01 0.96519 1.10951 0.97163 1.10077 0.78772 0.99369MLS000688437-01 1.03392 0.99294 1.10191 1.12359 1.19436 1.07625MLS001211976-01 1.1302 1.09895 1.19884 1.24128 1.2336 1.03242MLS002161350-01 1.11602 1.04912 0.98699 0.77831 1.17667 0.98443MLS001077207-01 1.20734 1.00773 1.14894 1.42157 1.06141 0.9848MLS001209245-01 0.57286 0.68765 0.99794 0.96674 1.24547 1.11315MLS000737953-01 0.53393 1.00681 1.22632 1.24139 1.13455 0.96648MLS000552080-01 0.92859 1.0378 1.23976 1.21445 1.14993 0.96716MLS000737204-01 0.66817 0.8815 1.11988 1.0331 1.17335 1.04251MLS000579238-01 0.77083 0.856 1.10409 1.01011 1.10886 0.9507MLS001181671-01 0.98779 0.98542 1.21954 1.33701 0.90946 1.07118MLS001167424-01 0.97794 1.0277 0.7848 1.36476 0.95956 1.04607MLS000094770-01 0.71187 0.81748 0.82301 1.31662 1.00014 1.13971MLS001123810-01 0.96938 0.95147 1.11589 1.41658 0.9657 0.97401MLS000532078-01 1.04337 1.07767 1.11359 1.239 1.07805 1.0727MLS000585616-01 0.96223 1.03062 1.14727 1.58056 0.95754 0.99906MLS000553673-01 1.13447 1.08812 1.16112 1.30384 0.89228 0.87494MLS001175592-01 1.04727 0.64453 1.03403 0.69108 0.986 0.96848MLS001033255-01 1.03969 0.86833 0.99699 1.21268 0.95257 0.96782MLS000733703-01 0.894 0.9957 1.14714 0.87792 1.12841 1.06336MLS001096269-01 0.91223 1.13461 1.11864 1.30716 1.08849 0.99921MLS001162872-01 0.88763 1.03393 1.03106 0.83691 1.05081 1.12992MLS000584511-01 1.04063 1.00352 1.16335 1.2189 1.05102 1.0682MLS001166325-01 1.0054 0.95925 1.06467 1.11151 1.04905 1.05671MLS000122180-01 0.94955 0.9557 0.74557 1.02716 0.93405 1.00577MLS001157804-01 1.18827 1.06942 1.08307 1.03088 1.01331 1.04505MLS000693729-01 1.05588 0.9682 1.14413 1.11873 0.77074 0.82188MLS001220669-01 1.01145 1.00318 1.03797 1.0117 0.97798 0.97225MLS001214461-01 0.88438 1.08667 0.94574 1.07181 0.95864 1.20656MLS000081838-01 1.03597 1.22502 0.87042 0.90024 1.01752 0.74245MLS002402866-01 0.92366 1.07525 1.00041 1.181 1.25798 1.09045MLS001200980-01 0.9712 1.01483 1.21582 1.12144 1.02235 1.0669MLS001174719-01 0.73741 0.97712 1.03422 1.12778 1.00054 1.13473MLS001175449-01 0.80412 1.15115 0.93663 1.15584 1.01599 1.1188MLS001172577-01 0.73236 1.14005 1.10122 1.13147 1.0349 1.12157MLS001122792-01 0.85223 1.06818 0.6414 1.10175 1.03173 1.19222MLS001216405-01 0.87057 0.93866 0.96422 1.12433 0.99268 1.12759MLS000673766-01 0.87173 0.82722 0.92763 0.89603 0.98625 0.96802MLS000912614-01 0.78553 0.77496 1.05484 1.10265 1.02407 1.04186MLS001194551-01 1.28081 1.20915 1.09418 1.04262 1.01038 1.0169MLS001214704-01 1.15847 1.05612 1.10982 1.07011 1.11315 0.97637MLS001219159-01 0.95074 0.94742 1.06395 0.80696 0.99654 0.861MLS001060549-01 0.91344 0.93911 1.11095 0.97834 1.07101 0.81811MLS000850824-01 1.20693 1.14342 1.0727 0.98997 1.09142 0.90829MLS000879190-01 0.8486 1.08434 0.91319 1.05175 1.13391 0.96497MLS001163140-01 1.01076 0.96509 1.1264 0.92815 1.03471 0.92359MLS000046123-01 0.92032 0.92679 0.86499 0.89047 0.93893 0.98024MLS000086970-01 0.88711 0.93125 0.96054 1.086 0.92199 0.95281MLS000388722-01 0.9404 0.80013 1.04165 1.03216 0.93686 0.92252MLS000676974-01 0.54473 0.97579 0.59939 0.70092 0.90205 0.91712MLS000772580-01 0.85346 0.94129 0.94737 1.32302 0.89986 0.85101MLS000698686-01 0.97223 0.6986 0.96603 1.01328 0.61586 0.88382MLS001162337-01 1.01442 1.16958 0.97377 1.00937 0.88495 0.69861MLS001204005-01 1.15931 0.81814 1.01778 0.76301 0.98694 0.90311MLS000721525-01 1.04717 0.89679 0.98056 0.66934 0.91358 0.66465MLS000525404-01 1.34722 1.19322 1.09097 1.11785 1.04083 1.04334MLS000772194-01 0.76515 0.99042 1.07217 0.96542 0.91916 0.97818MLS000775793-01 0.9508 0.99649 0.62239 0.87223 0.88326 0.93913MLS000710130-01 1.06401 1.04449 0.64515 0.61831 0.85241 0.98109MLS001146463-01 0.84238 0.84654 0.75138 1.21637 0.80315 0.88271MLS000712769-01 0.98169 0.92193 0.90375 1.2524 0.84283 0.97463MLS000334464-01 0.66588 1.12731 0.99178 1.00153 0.88028 0.98611MLS000862690-01 1.0763 1.0131 0.94809 0.59412 0.95764 1.12516MLS001179267-01 0.51107 0.85888 0.55899 0.89079 0.8787 0.94697MLS000683174-01 0.98644 1.08147 0.89073 1.00731 0.91651 1.14461MLS000913052-01 0.82866 0.97247 1.01326 1.05486 0.90137 1.07299MLS001080869-01 0.98021 0.98618 0.92569 0.97397 0.95744 1.01118MLS000332693-01 0.92516 0.97088 0.97983 1.09234 0.96309 1.0441MLS001141113-01 0.812 0.88742 0.92937 1.0538 0.85387 1.03774MLS001176611-01 0.98456 0.96653 0.93014 1.06623 0.66708 0.95057MLS001202627-01 0.23353 0.93939 0.81243 1.02515 0.76915 0.96869MLS000765108-01 0.7072 0.9224 0.60883 1.06063 1.01958 0.87214MLS000937079-01 0.91968 1.14203 0.8882 0.97562 0.63721 1.0515MLS001215742-01 0.96872 0.83842 1.15548 0.98031 1.10449 1.01971MLS001217045-01 1.05145 1.11523 1.15422 0.94 1.15574 1.00142MLS001196946-01 0.28319 0.54647 0.94694 1.00195 1.11272 0.99416MLS001216714-01 0.67688 0.706 1.3235 1.1023 1.17442 1.02706MLS000772430-01 1.04803 1.25987 1.25362 1.04073 1.2757 1.03466MLS000693370-01 1.245 1.15055 1.28243 1.15793 1.13104 1.10635MLS000769322-01 1.019 1.06721 1.16504 1.1195 1.05468 0.96093MLS000721030-01 1.20547 1.1412 1.2344 1.12136 1.05053 0.97569MLS001176897-01 1.16313 1.14489 1.19416 1.02235 1.07267 0.95773MLS000774940-01 0.87802 1.08429 0.99082 1.00988 0.97774 0.9469MLS001030746-01 1.06708 0.98862 0.94961 1.03058 0.96919 1.04669MLS003126425-01 0.52076 0.73725 1.6782 1.42164 1.20322 1.10413MLS001217697-01 1.11655 1.12294 1.19743 1.16037 0.96777 1.20389MLS000516719-01 1.17505 1.10738 1.18354 1.20263 1.0239 0.86182MLS001165323-01 1.08827 1.05423 1.15283 1.166 0.96267 0.94685MLS001220803-01 0.96218 1.0147 1.07507 1.17995 0.97031 0.93451MLS001163121-01 1.09034 1.07048 1.17333 0.93955 0.98945 1.05191MLS001060561-01 0.93292 0.85568 0.99768 0.98322 0.93293 0.80472MLS001139515-01 1.01273 1.12444 0.94878 1.01346 1.00945 0.9636MLS001149811-01 0.54317 0.84563 0.90865 0.94337 0.99933 0.95702MLS000773700-01 0.47196 0.98949 0.95155 0.71177 1.01875 1.00839MLS001177045-01 0.96779 1.10985 1.0851 1.02356 0.81454 1.04675MLS000693747-01 1.0456 1.12188 0.91107 1.43271 0.71944 1.06136MLS001175556-01 0.75145 0.90095 0.90127 1.09994 1.02814 0.94901MLS001175473-01 0.79712 1.04117 0.99773 0.8872 0.91773 0.95559MLS002156278-01 1.02456 0.97186 0.99244 0.69084 0.9637 1.00503MLS000707281-01 1.14087 1.15567 0.75052 1.05935 0.65371 1.01094MLS000591198-01 0.70144 0.98525 0.85477 1.00046 0.88105 1.01695MLS000714175-01 1.02244 1.11863 1.01289 1.13111 1.06141 1.08575MLS002163386-01 1.01995 1.06351 1.06415 1.11206 1.04751 1.10384MLS000761297-01 1.09327 1.04516 1.03348 1.15364 1.02167 1.0906MLS002245351-01 1.03102 1.06127 1.042 1.10724 0.94631 1.04658MLS000718886-01 0.97849 0.93799 0.99814 1.06782 0.95909 0.98188MLS002156485-01 1.15603 1.05728 1.0687 1.2005 0.98067 1.0551MLS001140657-01 0.97076 0.9948 0.99812 1.08308 0.95059 1.03407MLS002157024-01 1.07674 1.18645 0.93233 1.12188 0.88418 1.01348MLS000721730-01 1.07097 0.87695 1.02458 0.85627 1.06447 0.91949MLS000705922-01 1.03921 0.90721 0.95439 0.85107 1.13665 0.89838MLS000724709-01 0.97675 0.885 1.00234 1.04022 1.46739 0.99608MLS002161757-01 1.14604 0.97951 1.06023 1.09241 1.06958 0.8459MLS002164687-01 1.0594 1.01681 1.36762 1.23385 1.00235 0.85316MLS001060533-01 1.11677 1.34224 1.51976 1.31756 1.20754 0.94638MLS000685139-01 1.26695 1.10202 1.24049 1.12834 0.92023 0.87033MLS001217286-01 0.5964 1.09526 1.21873 1.19269 0.99106 0.76168MLS001221619-01 1.03393 0.86027 1.01242 0.94404 0.95568 0.82601MLS001219621-01 0.45369 0.648 0.86159 0.8883 0.86027 0.9394MLS001166156-01 0.44609 0.82177 0.75916 1.07035 0.85209 1.00131MLS000534926-01 0.85648 1.33769 0.9622 1.00584 0.94445 1.02592MLS000548725-01 0.22187 1.03126 0.84619 0.9206 0.84182 0.86975MLS000374261-01 0.44896 0.42298 0.92885 1.13089 0.83858 0.81441MLS000123454-01 0.88519 0.68413 1.08253 1.04655 0.89626 1.06673MLS000625140-01 0.65137 0.67934 0.91312 1.11353 0.84537 0.84836MLS001214443-01 1.08326 1.15407 1.19298 0.94422 0.90181 0.82616

TABLE 11 CMV-LACZ in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000591667-01 1.0593 0.8030 1.1592 1.1587 0.7850 0.9491MLS000568234-01 0.7322 0.8819 0.9894 0.9043 0.9372 0.9556MLS000689562-01 1.0619 0.9919 1.0858 0.8060 0.7409 0.8453MLS000732652-01 0.7856 0.9374 1.1235 1.1327 0.7744 0.6700MLS001240181-01 0.8907 1.0367 1.1645 0.8408 1.0441 0.9550MLS001211139-01 0.8744 1.0659 0.8608 0.9500 0.7347 0.9691MLS001030268-01 0.5556 0.7467 1.1787 1.1347 1.1550 1.0809MLS000912699-01 0.5204 0.8826 1.0462 0.9111 0.8744 0.7147MLS001125260-01 1.2270 1.0563 0.9131 1.0114 1.0584 1.1581MLS000717689-01 0.8996 1.0407 1.0936 1.1129 1.0203 1.0125MLS001197665-01 1.0267 0.9556 0.8403 0.9214 0.8347 0.8584MLS001075922-01 1.3037 0.9881 1.2361 1.6332 1.3028 0.9434MLS001124046-01 1.0807 0.9881 0.8501 0.9307 1.2650 0.8409MLS001197220-01 1.2844 0.9567 1.1383 0.9355 1.1569 1.2028MLS001221318-01 1.5641 1.0181 0.9955 1.0999 1.0538 0.8528MLS000947910-01 1.0500 1.0870 1.0379 0.9452 0.9250 1.0094MLS001215795-01 0.8256 0.8196 0.8628 1.0974 1.1375 0.8003MLS002163670-01 1.4715 0.8611 0.9725 0.8671 0.8738 1.1806MLS001200149-01 1.1552 0.9363 1.0523 0.9489 0.9203 1.1234MLS001359861-01 1.0330 0.9074 0.8064 0.9009 0.9184 0.9444MLS000710669-01 0.8778 1.1319 1.0823 1.0325 0.9975 1.0969MLS001035690-01 0.9178 1.3585 1.2020 0.9910 1.0406 0.9819MLS001030621-01 0.9063 1.0307 1.1844 1.0494 1.0531 0.7081MLS001083082-01 0.8144 0.9615 1.1713 1.0409 0.9234 0.8591MLS000045588-01 1.2167 0.8056 1.0611 1.0245 0.8372 0.7647MLS001216939-01 1.0644 1.1863 0.9452 1.0913 1.1134 1.0991MLS001163859-01 0.8104 0.7593 1.1812 1.2009 1.1684 1.1113MLS000683232-01 0.8237 0.6119 0.5224 0.5638 0.7131 0.8043MLS001170856-01 0.8389 0.8881 1.0004 0.7938 0.9857 0.7337MLS002667707-01 1.1226 1.0044 1.0803 1.1897 0.6763 1.1434MLS001200665-01 0.9937 0.7152 1.0036 0.9398 0.9783 1.1043MLS002161853-01 0.9348 1.0922 0.9436 0.8819 0.8734 1.0131MLS002163101-01 1.1556 0.9644 1.0313 1.0262 1.0317 0.9991MLS000062431-01 1.0993 1.0467 1.1632 0.9848 1.1631 0.9557MLS000028160-01 0.8600 0.8967 1.2759 0.9378 0.9029 1.0663MLS002248819-01 1.0270 1.7330 1.1575 1.2604 1.2206 0.9206MLS000080654-01 0.8815 1.0174 0.9345 1.0703 1.4314 1.1851MLS000760876-01 0.8504 0.9396 0.8973 0.8293 1.0546 0.9069MLS000677675-01 1.1578 1.0763 1.0373 0.9905 1.1214 1.0343MLS000113985-01 0.1841 0.2126 0.9187 1.0836 1.0049 0.9809MLS001182368-01 1.1615 1.2322 0.9225 1.0528 0.8783 0.9571MLS001212882-01 1.0326 1.3230 1.0086 0.9792 1.1291 1.1409MLS001004364-01 1.0441 1.0263 0.9011 1.0438 1.0643 0.9117MLS000736846-01 1.0174 1.3459 1.1101 1.0269 1.0386 1.1009MLS001098105-01 0.8704 1.0381 0.9421 0.9282 0.8054 1.4689MLS000678673-01 0.9656 0.8674 0.8991 1.0561 1.1914 1.2106MLS000925023-01 0.3278 0.5504 0.8397 0.9762 0.9629 1.0703MLS001212319-01 1.0259 1.2056 0.8600 1.0840 1.2040 0.9129MLS000779126-01 0.7441 0.9944 0.7610 0.9637 0.9994 0.9751MLS000948055-01 0.5522 0.5407 1.0993 1.0787 0.9674 0.7540MLS000110418-01 0.7141 0.8926 0.9455 1.0913 0.9989 0.8871MLS000693704-01 1.0567 0.7996 0.9977 0.9522 1.1157 0.8989MLS001225512-01 1.0111 0.8237 1.4266 1.1655 1.0843 0.9597MLS001006798-01 0.8263 0.9622 1.0189 1.1509 1.0326 0.8334MLS000711491-01 0.7800 1.5063 1.0199 0.9088 1.1060 1.0889MLS000582947-01 0.8819 0.7811 1.0067 1.1273 0.8991 0.9691MLS000531177-01 0.9422 1.2952 0.8958 1.0150 1.5065 1.0785MLS001202389-01 0.5081 0.6115 0.8920 1.0084 1.1108 1.0237MLS000536064-01 2.4896 2.5396 1.3388 2.0402 0.7412 0.9105MLS000586245-01 0.7674 0.7726 1.0505 0.9258 0.8985 0.9034MLS001061374-01 1.1037 0.9744 0.9793 1.0601 1.1702 0.7117MLS000675441-01 0.7437 0.8656 0.8690 0.8800 0.9289 1.0332MLS001200396-01 0.9011 0.7252 1.2107 1.0212 0.9157 1.0489MLS001165937-01 0.4007 0.4526 0.7441 0.8816 0.7788 0.7295MLS000325736-01 1.0537 1.0111 1.0696 0.9758 0.9135 0.7862MLS001215357-01 0.4719 1.2467 1.0378 0.8575 1.2686 1.1560MLS000588210-01 0.9000 1.0307 0.9316 0.7808 0.9782 1.2908MLS000764729-01 0.7504 1.1200 0.8880 1.0259 0.9520 0.9865MLS000689492-01 1.0026 1.0141 0.8653 1.0863 1.0252 0.9363MLS001000299-01 1.0107 1.1522 0.8248 0.9901 1.2745 1.4188MLS000393762-01 0.4467 0.3793 1.0198 1.0114 0.9351 0.9560MLS001130011-01 0.8311 0.7837 1.1138 1.0011 0.7695 1.1702MLS001229477-01 0.8800 1.0674 1.1311 1.1078 1.1345 1.0283MLS000707378-01 0.6548 0.8667 0.8072 0.8309 1.0883 1.0175MLS000573208-01 0.9289 0.6626 0.8678 1.1030 0.9594 1.0197MLS001167281-01 0.7467 0.9256 1.1951 0.9684 1.1289 1.0114MLS000053342-01 0.9930 0.9811 0.8785 1.0865 1.0397 1.0289MLS002171615-01 0.6726 1.2159 0.9905 0.9273 1.3505 1.0498MLS001005712-01 1.0230 1.0241 0.9125 0.8934 1.2385 1.0188MLS001176153-01 1.0763 0.9322 0.9140 0.8976 1.4588 1.2729MLS000735021-01 0.9767 1.2130 0.9907 0.9756 1.1646 0.8683MLS000767397-01 1.1433 0.8485 0.9687 0.9910 1.0646 1.2748MLS001196572-01 1.0778 1.0767 0.9456 1.0605 1.3172 1.0105MLS000393966-01 0.9156 1.0411 0.8087 1.1744 1.0625 0.9249MLS001034810-01 0.7030 1.1193 0.9459 1.0948 0.9538 0.9698MLS001165394-01 0.9570 0.8663 0.9792 1.0941 1.0015 1.0655MLS000089464-01 0.5274 0.7500 1.1969 0.9372 0.9160 0.9117MLS000698617-01 1.0356 1.1670 1.0603 1.1399 0.9852 1.3409MLS001175021-01 1.0907 1.1070 0.9395 0.9282 1.0729 1.1772MLS001166758-01 0.3481 0.6126 1.0661 1.1998 1.0175 0.9357MLS001008109-01 0.7619 0.8730 1.2178 0.9228 1.1138 0.9542MLS001181936-01 0.6130 0.7863 0.8296 0.7292 0.9273 0.8487MLS000560266-01 1.0484 1.1095 0.9991 0.9405 0.8123 0.9030MLS001215074-01 1.1432 1.1277 1.0294 1.1754 0.7800 0.7877MLS001215123-01 1.0098 1.2639 1.1943 1.0888 0.8837 1.0530MLS001033255-01 0.7905 1.1919 1.0219 0.8766 1.0723 0.7590MLS001160611-01 1.0389 0.8144 1.0340 1.0117 0.8157 1.2070MLS001006302-01 0.7646 0.9502 1.0767 0.8911 0.7663 0.9343MLS001123876-01 0.7333 0.9618 0.9871 0.9251 1.0263 0.8260MLS001122698-01 1.0639 1.0404 0.8608 0.7759 1.1003 1.1513MLS000755214-01 0.8172 1.0835 1.0994 0.9281 1.1650 1.0190MLS000731285-01 0.6572 0.9582 0.8436 1.0851 1.0853 1.1747MLS000776409-01 0.2105 1.3344 0.9052 0.8929 0.9103 1.1220MLS001221908-01 0.6789 0.9726 0.7908 0.9357 0.7607 0.9663MLS000419286-01 0.6295 0.9368 0.8022 0.9701 0.7960 0.9960MLS000554416-01 0.8611 1.1077 1.1123 0.8594 1.3983 1.1310MLS000073150-01 0.9000 0.9491 0.9954 0.8193 1.8970 1.0753MLS000663185-01 0.9347 0.9211 0.8567 0.9688 1.1477 1.1390MLS001078811-01 0.9014 0.8393 0.6873 0.7927 1.2290 1.2397MLS002694363-01 0.5698 1.0123 1.0392 0.7616 1.2840 1.1110MLS000689218-01 1.6337 1.2835 1.0813 0.8689 1.2273 1.1977MLS001215294-01 0.4116 0.3302 1.3412 0.8138 1.2817 1.2983MLS001183575-01 0.8856 1.2825 0.7762 0.8972 1.2440 1.1603MLS000393567-01 1.1319 1.0175 0.8148 0.6593 1.1373 1.2117MLS000546316-01 1.5049 1.3463 1.2440 0.8090 1.1373 1.0623MLS000912258-01 0.8621 1.1074 1.0171 0.9071 1.1693 1.1073MLS000850522-01 1.0091 0.8358 0.8671 0.8809 1.3763 0.8880MLS001197779-01 1.1267 0.9846 0.6490 0.7604 1.2563 0.8987MLS001095705-01 1.1723 1.1400 0.7229 0.6476 1.2293 1.2060MLS000912562-01 0.8747 1.0302 0.6246 0.7103 0.9150 0.8680MLS000092785-01 0.3235 1.1004 0.7351 0.9882 1.1653 0.8600MLS000418650-01 1.1323 1.1523 0.8344 1.0080 0.9533 0.9237MLS000860966-01 0.7600 0.6232 0.8424 0.7918 1.0900 0.9370MLS001180929-01 0.6163 0.9811 0.8753 0.8251 0.9288 0.8571MLS000564564-01 0.8844 1.0422 0.9091 0.9399 1.0092 0.9117MLS001164432-01 1.1289 1.1059 1.3183 1.1387 0.7842 0.9600MLS000621451-01 1.1926 1.2493 1.3023 0.9255 1.1579 1.0388MLS000078709-01 0.4830 0.6919 1.0163 0.9270 0.7008 0.8167MLS000688267-01 1.0774 1.1407 0.8935 0.9129 1.1579 1.0950MLS001006516-01 0.6589 0.4956 0.9076 0.9829 0.8858 1.0038MLS001095033-01 0.9811 0.9359 1.0269 0.9043 0.9179 0.9488MLS000072290-01 0.2915 0.3911 1.3303 1.4390 0.9000 1.0579MLS000036988-01 0.6578 0.8796 1.0435 0.9702 1.0846 1.0767MLS001217935-01 0.5381 0.7859 1.0465 1.0175 1.1167 1.1729MLS000777780-01 0.9463 1.3393 1.0126 0.9893 0.9063 0.8779MLS001174740-01 0.4578 0.4293 0.9490 1.0046 0.9721 1.2750MLS000392555-01 1.2007 0.9559 0.9799 0.9342 0.7638 0.8954MLS000693795-01 0.8730 0.8526 0.9207 0.9554 1.1104 0.9100MLS000684034-01 0.3100 0.5374 1.0568 1.2045 1.1492 1.2525MLS001172822-01 1.2767 0.9285 1.0904 1.1375 1.0617 0.9304MLS000052969-01 0.6800 1.1522 1.0444 0.9976 1.0529 0.8950MLS001217212-01 0.9459 1.1137 1.0626 1.0711 0.9179 1.1804MLS001004864-01 0.9933 1.2930 1.0417 1.2276 0.9288 1.2650MLS001116535-01 1.1263 1.1815 1.0580 1.0944 1.1779 1.0167MLS001165424-01 1.2074 1.2730 1.0078 1.0607 0.9796 1.1754MLS001116079-01 0.9078 1.0448 1.0228 0.9306 1.0492 1.0950MLS001198271-01 1.2089 0.9944 0.8516 1.2105 1.0883 1.0204MLS001167798-01 1.1452 1.2196 1.1494 1.1822 0.7750 1.0342MLS000710288-01 0.9063 1.1193 1.2515 1.0737 0.9325 1.0338MLS000734270-01 0.9548 1.0122 0.9468 0.9337 0.8379 0.9296MLS000858981-01 0.9696 0.9985 0.9009 1.1687 0.9696 0.9229MLS000698826-01 1.2519 1.2944 0.9013 0.8824 0.9717 0.9942MLS001000874-01 1.1100 1.0556 1.0295 0.8886 1.0442 1.1029MLS000682750-01 1.0404 1.1433 0.9934 0.8717 1.0467 0.9679MLS001090787-01 0.5907 0.7533 0.8594 0.9700 0.9754 0.9196MLS002636056-01 0.8270 1.1173 1.2571 1.0394 0.9021 0.9392MLS002170630-01 0.7387 0.8233 0.9628 0.9890 0.8054 0.9954MLS002162890-01 0.9507 0.7603 0.9939 0.9701 0.8713 0.8392MLS001105912-01 0.3873 0.5323 0.9597 1.0003 1.0321 1.0725MLS001007892-01 0.4953 0.6390 1.1495 1.0511 0.8108 0.9338MLS000089748-01 0.4437 0.3140 0.9905 1.0665 0.8592 1.2129MLS000912726-01 1.0643 1.0257 1.0325 1.0182 0.9692 1.1004MLS000086970-01 0.8880 1.0197 0.9068 0.8994 1.0504 0.9925MLS000420298-01 1.1930 0.9807 1.0482 0.8414 0.9383 1.2808MLS001147478-01 0.8747 1.0513 1.2999 1.2105 0.8808 1.0721MLS000090135-01 0.3653 0.6017 0.9296 0.9731 0.9421 1.0896

TABLE 12 CMV1:AB305V-LACZ in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000591667-01 0.8800 0.8929 1.0152 1.0824 1.0726 0.8600MLS000568234-01 0.9141 0.9918 0.9074 1.0764 1.2622 1.3844MLS000689562-01 0.9788 0.9824 0.8105 1.0300 0.8652 0.9474MLS000732652-01 1.0329 0.8518 1.0697 1.0614 0.8859 1.1615MLS001240181-01 1.1376 1.0976 0.9955 1.0895 1.0511 0.9519MLS001211139-01 1.0224 0.8259 1.0196 1.1755 0.8948 0.9281MLS001030268-01 0.9506 0.9365 1.1128 1.1318 1.1711 0.8830MLS000912699-01 0.5365 0.9235 1.1310 0.9419 0.9859 0.8089MLS001125260-01 0.9212 1.0000 1.0994 0.9614 1.0993 1.0074MLS000717689-01 0.9129 0.9576 0.9569 0.9686 1.0956 0.8415MLS001197665-01 1.0741 1.0047 1.2593 0.9771 0.8756 0.9415MLS001075922-01 0.8388 1.0282 1.2173 1.2947 1.0533 1.0222MLS001124046-01 0.9812 1.0906 1.0202 0.8395 1.0156 1.0193MLS001197220-01 1.0271 0.9659 0.9289 0.9682 0.9356 0.8896MLS001221318-01 0.8576 1.2212 0.9944 1.1337 1.4059 1.0037MLS000947910-01 1.0082 1.0165 0.8930 1.2332 0.8348 1.0333MLS001215795-01 1.1035 1.3929 1.0250 0.9309 1.0207 0.9363MLS002163670-01 1.1235 1.1659 0.8898 0.9788 1.2044 0.9237MLS001200149-01 0.9424 0.8318 0.8176 0.9974 0.9933 0.8378MLS001359861-01 0.9412 0.9976 1.1230 0.9547 0.9533 0.8978MLS000710669-01 1.1824 0.8682 1.2372 0.9690 0.8881 0.9341MLS001035690-01 1.0976 1.1976 1.1317 1.1552 0.8644 1.0474MLS001030621-01 1.0141 0.9024 1.0546 0.9872 0.7970 0.7844MLS001083082-01 1.1165 1.0282 0.9776 0.9984 1.0326 0.9615MLS000045588-01 0.9424 1.0494 0.9776 1.2294 0.8607 0.9059MLS001216939-01 0.9659 1.1518 1.2568 1.1826 0.9748 1.0311MLS001163859-01 1.2482 1.0965 1.1727 1.0856 1.1326 0.8756MLS000683232-01 0.8365 0.8835 1.0086 0.8743 0.7752 0.8200MLS001170856-01 0.8753 1.1518 1.0562 1.0074 0.9105 1.1095MLS002667707-01 1.1071 1.1576 1.0227 0.9449 1.0590 0.8905MLS001200665-01 1.2706 1.1800 0.9662 1.1274 0.9695 1.1543MLS002161853-01 1.0647 1.2341 0.9103 1.1122 0.8352 1.0524MLS002163101-01 1.5447 0.9212 0.8407 0.8302 1.1400 1.0533MLS000062431-01 1.2047 0.8812 0.9690 0.9004 0.9743 0.9133MLS000028160-01 1.1976 1.0471 0.9249 1.0348 1.0362 1.0524MLS002248819-01 1.1929 0.9847 1.1534 1.1171 0.9505 1.0819MLS000080654-01 1.4259 1.1871 1.0399 1.0335 1.0086 1.1229MLS000760876-01 1.2565 0.9482 0.9789 1.1074 1.1010 1.0248MLS000677675-01 1.2494 0.9918 1.0091 1.0578 0.9962 0.8619MLS000113985-01 1.0482 1.0541 1.0241 1.0031 1.0638 1.0629MLS001182368-01 1.0318 0.9953 0.8690 0.8931 1.0619 0.8667MLS001212882-01 1.3024 1.1471 1.1133 1.0963 0.8705 1.0238MLS001004364-01 1.1824 1.2824 0.9480 0.8978 1.0095 0.9210MLS000736846-01 1.2788 1.1000 1.1737 1.0250 0.9714 0.8438MLS001098105-01 1.2553 0.9671 1.3183 0.9211 1.2543 0.9248MLS000678673-01 1.1835 0.9635 0.9367 0.7918 1.0286 1.1229MLS000925023-01 1.1106 1.1565 0.8859 1.1638 1.1333 0.7505MLS001212319-01 1.0706 0.9965 0.9253 0.9525 0.8152 1.1524MLS000779126-01 1.1671 0.9988 1.0467 1.0257 1.2162 0.9495MLS000948055-01 1.0753 1.2212 1.1022 1.2519 0.9638 1.0895MLS000110418-01 1.3647 1.0459 0.9599 0.9617 0.9000 1.0333MLS000693704-01 1.0600 1.1647 1.0812 1.1170 0.8695 0.8571MLS001225512-01 0.8871 1.2482 1.0077 0.9459 1.0381 1.0162MLS001006798-01 1.0976 0.9953 1.0267 1.2236 0.9648 1.0419MLS000711491-01 0.9929 1.1318 0.8645 0.9993 0.9095 0.9495MLS000582947-01 1.0247 1.0729 0.9127 0.9280 0.9610 0.8848MLS000531177-01 1.1522 0.9244 0.8917 0.8757 1.0411 1.1989MLS001202389-01 0.9311 1.0900 1.1312 1.0432 1.1821 1.1558MLS000536064-01 1.0233 1.2344 1.1027 0.8894 1.0389 0.9705MLS000586245-01 1.2656 1.2267 1.1338 0.9511 1.0800 1.0611MLS001061374-01 1.0489 1.1689 0.8498 1.1237 1.1000 1.2568MLS000675441-01 0.9822 0.9578 1.0337 1.0460 1.1032 1.0074MLS001200396-01 0.9733 0.9644 0.7738 0.8987 0.8442 0.9137MLS001165937-01 0.7222 0.7678 0.7827 0.8619 0.9663 0.9305MLS000325736-01 0.9889 0.8244 0.9324 0.9630 0.8968 0.9263MLS001215357-01 1.0667 1.3622 1.1192 1.1407 1.1400 1.1547MLS000588210-01 1.1656 1.1322 1.0815 0.9182 1.0853 1.1263MLS000764729-01 1.1900 0.9656 0.9634 1.0779 1.1432 1.0358MLS000689492-01 0.9267 1.0444 1.0940 1.0504 1.2400 1.0853MLS001000299-01 1.0744 0.9544 0.9029 1.0658 1.3158 0.6411MLS000393762-01 1.0578 1.0800 1.0049 1.0005 0.9326 0.8653MLS001130011-01 1.0156 0.8367 0.8998 0.9057 1.0211 0.9358MLS001229477-01 0.9611 0.9622 0.9587 0.9250 1.1095 1.0126MLS000707378-01 0.9167 0.9689 1.0300 0.9294 1.3937 1.0537MLS000573208-01 0.9478 0.9633 0.8913 1.1765 1.3021 0.9547MLS001167281-01 0.9722 1.1089 0.9727 0.8948 1.0789 1.0663MLS000053342-01 1.1344 1.3044 0.9187 0.9033 0.9863 0.9947MLS002171615-01 1.0711 1.0111 0.9582 0.9989 0.7874 0.9863MLS001005712-01 1.3167 1.1133 0.8075 0.9623 1.0379 1.0684MLS001176153-01 1.0878 1.0256 0.9388 0.7944 0.9832 0.8316MLS000735021-01 1.0778 1.0378 0.9616 0.9548 1.0221 1.0105MLS000767397-01 1.0067 1.0144 0.9519 0.9425 1.0368 0.8737MLS001196572-01 1.1422 1.1456 1.0223 0.9853 0.9379 0.9632MLS000393966-01 1.1167 1.0378 1.1877 0.8958 1.3516 0.9358MLS001034810-01 1.1344 1.0578 1.0965 0.9578 1.7389 1.1000MLS001165394-01 0.9544 1.0344 0.9045 0.9589 1.1558 0.9232MLS000089464-01 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0.93728 1.00213 0.93957MLS001139515-01 0.96009 0.92368 0.76043 0.94043 MLS001149811-01 1.650441.12193 1.04766 0.9566 MLS000773700-01 0.40921 0.92895 1.1166 1.12553MLS001177045-01 1.11798 1.05833 1.11021 0.92383 MLS000693747-01 1.042980.9693 0.95234 0.99617 MLS001175556-01 1.10263 0.91579 1.05277 1.11277MLS001175473-01 1.06579 0.95921 0.85872 0.89617 MLS002156278-01 1.031140.96667 1.09574 0.86936 MLS000707281-01 0.95263 0.94737 0.92553 0.87064MLS000591198-01 0.45307 0.83377 0.93191 0.96809 MLS000714175-01 0.971051.12412 1.09745 0.93191 MLS002163386-01 1.06623 0.94649 1.19617 0.97489MLS000761297-01 0.88289 0.99298 0.81064 0.99106 MLS002245351-01 0.93071.00921 1.00128 0.83064 MLS000718886-01 0.83553 0.87982 0.98043 0.97872MLS002156485-01 0.94825 1.11491 0.95234 0.94681 MLS001140657-01 1.0751.05044 1.04596 1.05319 MLS002157024-01 0.83333 1.05921 0.85532 1.14638MLS000721730-01 1.03067 0.93111 0.92069 0.85043 MLS000705922-01 0.994670.93022 0.9306 0.87759 MLS000724709-01 1.12533 1.05378 0.88276 0.84224MLS002161757-01 1.256 1.15289 1.10216 0.91897 MLS002164687-01 1.113330.968 0.92198 0.7625 MLS001060533-01 0.68711 0.77156 0.77888 0.83578MLS000685139-01 0.93867 1.03111 1.03621 0.93621 MLS001217286-01 0.360440.39911 1.22672 0.95991 MLS001221619-01 1.78133 1.51333 1.32069 1.17155MLS001219621-01 1.29022 1.132 1.06293 0.94914 MLS001166156-01 0.522670.77956 0.91983 0.87069 MLS000534926-01 0.844 0.95467 0.96897 0.97241MLS000548725-01 0.332 0.87289 1.12629 0.97888 MLS000374261-01 0.503560.448 0.8319 0.82457 MLS000123454-01 1.05467 0.87956 0.93233 0.93534MLS000625140-01 1.016 1.10667 1.03621 0.91724 MLS001214443-01 0.902220.95067 1.07241 0.97371

TABLE 13 Burkin Assay in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000763074-01 0.60851 0.65857 1.13427 0.843 1.06668 1.19903MLS000564846-01 0.75904 0.74063 0.87038 0.80671 0.84376 0.83593MLS001202366-01 0.24561 0.48734 0.74576 0.87622 0.96981 1.00673MLS001182278-01 0.84246 0.76628 1.17046 1.06287 1.0742 1.21321MLS001196427-01 0.87626 0.81017 0.97767 0.93838 1.03303 1.01962MLS001202425-01 0.34448 0.74647 0.86448 0.96729 1.21932 1.2214MLS001214276-01 0.02694 0.20219 0.32354 0.62344 1.22246 1.32758MLS000682748-01 0.24723 0.46691 0.765 0.89229 1.16212 1.259MLS000834756-01 1.08092 1.06424 1.17192 1.22657 1.09843 1.05646MLS001202402-01 0.32496 0.64813 1.08015 0.92555 0.90338 1.05088MLS001214264-01 0.23942 0.78788 0.70087 0.74298 0.84644 0.8908MLS000391588-01 0.30047 0.78036 0.81758 0.91191 1.01537 1.06962MLS001013431-01 0.33625 0.45216 0.53107 0.70768 1.00039 1.20211MLS001163848-01 0.14441 0.29703 0.31406 0.48542 0.89062 1.00231MLS000327715-01 0.57757 0.90154 1.02899 1.14772 1.09422 1.37572MLS001214300-01 0.02593 0.18472 0.35298 0.80412 0.93656 1.21103MLS000834755-01 0.79619 1.19253 1.15647 1.08036 1.07782 1.27365MLS001163860-01 0.23471 0.17222 0.49218 0.57932 0.84566 0.94942MLS000710233-01 0.17433 0.17232 0.56369 0.80665 1.15918 1.17028MLS002702497-01 0.04886 0.05588 0.18885 0.17496 0.55454 0.78041MLS000546982-01 0.88835 1.05267 1.07821 1.27207 1.15498 0.87993MLS001197838-01 0.86478 0.89105 1.04838 1.05318 1.19792 1.25617MLS001202354-01 0.42755 0.51598 1.07323 1.03576 1.02304 1.07717MLS001202330-01 0.64675 0.83048 0.93405 0.95578 1.02782 0.97384MLS001182377-01 0.61764 0.87441 0.90613 0.91503 1.04505 1.17882MLS000834757-01 1.02067 1.11712 0.96506 1.12452 1.00317 1.0102MLS001202634-01 0.93072 0.99395 1.31203 1.14099 1.10045 1.20777MLS001194544-01 0.50277 0.79143 1.14522 1.02036 0.99063 1.04429MLS000554109-01 0.89366 0.9611 1.09535 1.06742 1.04915 0.99289NCGC00263072-01 0.98868 1.10983 1.04886 1.01335 1.24172 1.27253NCGC00263071-01 0.53023 0.69803 0.78353 0.9185 1.13222 1.35429NCGC00263039-01 0.81077 0.99561 1.02563 1.02082 0.91594 1.1697NCGC00164631-03 0.01283 0.01408 0.01373 0.32241 1.03203 0.83726NCGC00179302-02 0.80668 0.84305 0.71633 0.60599 1.0859 1.06101NCGC00241113-01 0.9168 0.95936 0.86618 0.9062 1.05675 1.01086NCGC00094381-04 0.64658 0.77957 0.63974 0.7317 1.05336 1.03555NCGC00015546-04 0.14966 0.19368 0.21399 0.37707 0.92282 0.89229NCGC00094381-03 0.52027 0.6668 0.65793 0.73271 0.9952 0.93685NCGC00094381-05 0.75776 0.88787 0.7145 0.75321 0.98039 0.94327NCGC00015545-02 0.18236 0.30858 0.31898 0.59598 1.32546 1.17517NCGC00164631-04 0.01361 0.01476 0.08696 0.52898 1.01135 1.02287NCGC00094112-04 0.34285 0.45094 0.49426 0.57218 0.99267 1.01949NCGC00242500-01 0.01945 0.01841 0.58791 0.87045 1.07607 1.05199NCGC00015545-07 0.242 0.30684 0.56798 0.84629 1.12041 1.07252

TABLE 14 Burkin Assay in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000763074-01 0.26221 0.19215 0.90703 1.04266 1.12824 1.0274MLS000564846-01 0.75294 0.70065 1.07066 1.00195 0.95569 0.88356MLS001202366-01 0.51046 0.52721 0.91937 1.08888 1.09876 1.11392MLS001182278-01 0.64876 0.56532 1.41234 1.40178 1.11835 1.1969MLS001196427-01 0.93648 0.79418 1.13545 1.02154 1.01999 0.91943MLS001202425-01 0.35401 0.51782 0.80333 0.9499 1.20487 1.14828MLS001214276-01 0.07736 0.23625 0.69453 0.9019 1.19651 1.16185MLS000682748-01 0.62062 0.74301 1.08485 1.14662 1.21063 0.98015MLS000834756-01 0.91886 0.94946 0.9214 1.09476 1.18855 1.07853MLS001202402-01 0.37173 0.59069 1.0295 0.96293 0.89618 0.96279MLS001214264-01 0.71731 0.71648 1.05756 0.90726 0.89746 0.95761MLS000391588-01 0.66403 0.76307 0.93558 0.89942 0.95626 1.07272MLS001013431-01 0.70692 0.7376 0.85055 0.85067 1.0097 1.23758MLS001163848-01 0.48759 0.68784 0.74688 0.95866 1.04499 1.0964MLS000327715-01 1.11892 1.19159 1.11494 1.30229 1.02522 0.88377MLS001214300-01 0.31784 0.74468 0.87335 1.09139 1.03917 1.01853MLS000834755-01 0.81603 0.96865 0.98675 1.00334 1.23201 1.26329MLS001163860-01 1.20854 0.86198 1.13792 0.92233 0.94035 1.00928MLS000710233-01 0.62947 0.69332 0.86883 0.93307 1.033 1.11028MLS002702497-01 0.38389 0.45669 0.78137 0.76924 0.74901 0.84142MLS000546982-01 0.71115 0.85558 0.99622 1.14451 1.03799 1.04819MLS001197838-01 0.99815 0.86987 1.09145 1.00443 0.98105 1.00877MLS001202354-01 0.57849 0.5824 1.06858 0.95451 0.90225 0.84594MLS001202330-01 0.62071 0.63028 1.01709 0.91234 0.91736 0.97095MLS001182377-01 0.91488 0.86542 0.9389 0.93717 1.02095 1.01535MLS000834757-01 0.94642 0.99887 0.95369 1.02962 0.94297 1.01879MLS001202634-01 1.28943 0.94727 1.13854 0.9675 1.1156 0.99278MLS001194544-01 0.76105 0.754 1.06983 1.00203 0.9995 0.96112MLS000554109-01 1.04819 0.84256 0.98744 0.96115 1.05362 0.88389NCGC00263072-01 0.88975 0.93047 0.95374 0.96945 1.00581 0.98195NCGC00263071-01 0.65683 0.68367 0.77685 0.86036 0.89218 0.86806NCGC00263039-01 0.59697 0.74735 0.86326 0.98314 0.95574 0.96891NCGC00164631-03 0.07758 0.09031 0.18808 0.55055 0.94454 0.95119NCGC00179302-02 0.68712 0.68199 0.79125 0.81812 0.93769 0.93491NCGC00241113-01 0.63117 0.74575 0.76777 0.84894 1.10523 1.07764NCGC00094381-04 0.93082 0.76798 0.87417 0.83691 0.94176 0.8006NCGC00015546-04 0.79646 0.78811 0.82527 0.85291 1.05081 0.9443NCGC00094381-03 1.15097 0.94969 1.02446 0.90148 0.93337 0.8564NCGC00094381-05 1.13784 1.05028 1.31892 1.04722 0.93095 0.92337NCGC00015545-02 0.35192 0.49594 0.55162 0.69213 0.91177 0.91301NCGC00164631-04 0.08093 0.08218 0.21321 0.77386 0.99976 0.97606NCGC00094112-04 0.34997 0.68129 0.62896 0.7596 0.81588 0.74019NCGC00242500-01 0.21319 0.652 0.75336 0.94215 0.93914 0.99743NCGC00015545-07 0.49755 0.66539 0.74573 0.87208 1.08023 1.12579

TABLE 15 CMV-LACZ in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000763074-01 1.6644 1.2981 1.1966 1.1447 0.8256 0.8431MLS000564846-01 1.0885 1.2363 1.3619 0.9379 0.8172 0.9922MLS001202366-01 0.3322 0.7222 1.3214 1.0533 0.6431 0.7656MLS001182278-01 0.5959 1.0519 0.9778 0.9823 0.9491 0.7466MLS001196427-01 0.9563 0.7144 1.1811 0.9957 1.0356 0.7219MLS001202425-01 0.6233 0.9081 1.1386 0.8675 1.0919 1.0625MLS001214276-01 0.1781 1.2922 1.2231 1.3014 0.8731 0.9516MLS000682748-01 0.2419 0.3893 0.9959 1.0215 0.9666 0.9075MLS000834756-01 1.1889 0.8022 1.0917 1.2652 1.0422 0.8709MLS001202402-01 0.4819 0.7204 0.7598 0.8771 1.0106 1.0546MLS001214264-01 0.4856 0.3870 0.9233 1.3058 1.1383 1.0657MLS000391588-01 1.1119 1.4307 1.0968 0.9885 0.7411 1.0009MLS001013431-01 0.6670 0.5904 0.6673 0.8366 1.0640 0.9906MLS001163848-01 2.2215 2.1030 1.0762 0.9336 0.9620 1.1146MLS000327715-01 1.0648 1.3181 1.0060 0.9664 1.1731 1.2294MLS001214300-01 0.1793 0.4007 1.1047 0.7949 0.9403 1.0446MLS000834755-01 0.9133 0.9007 0.9834 0.9591 1.0400 1.0317MLS001163860-01 0.6488 0.3425 1.1417 0.9610 0.8230 0.9350MLS000710233-01 0.8779 0.8530 0.8509 0.8263 0.8263 1.0173MLS002702497-01 0.2779 0.2593 3.6076 2.6344 1.0213 0.8643MLS000546982-01 1.3937 0.9512 1.0785 0.9273 1.2640 1.0877MLS001197838-01 0.9437 1.1615 1.1118 1.0010 0.9954 0.7904MLS001202354-01 0.7437 0.8378 0.9170 0.9669 0.9521 1.2275MLS001202330-01 0.7619 0.9511 0.9082 1.0099 0.9325 0.7417MLS001182377-01 0.4170 0.4441 0.9294 0.9113 1.1646 1.1229MLS000834757-01 1.1026 0.8115 1.0942 0.9101 0.9300 1.0367MLS001202634-01 0.9217 1.0817 1.0990 1.1287 1.0071 1.0500MLS001194544-01 0.7383 0.8983 1.0363 0.9068 0.7213 0.8975MLS000554109-01 0.9650 0.9853 0.9457 0.9003 1.1863 1.1083NCGC00263072-01 1.2880 1.1540 1.1158 1.0035 0.9983 1.2383NCGC00263071-01 0.6430 0.7803 0.9908 1.2012 1.2813 1.0800NCGC00263039-01 0.9543 0.9740 0.9335 1.0522 1.1700 1.0746NCGC00164631-03 0.1767 0.1830 0.2763 0.7817 0.8658 NCGC00179302-021.2873 1.0247 1.6628 2.4679 1.1867 NCGC00241113-01 3.3097 2.7827 1.13521.1634 1.4142 NCGC00094381-04 0.9697 0.9330 0.9770 1.1366 1.0763NCGC00015546-04 0.5873 0.8083 1.2439 1.4889 1.1442 NCGC00094381-031.0253 0.7777 1.0089 0.8667 0.9504 NCGC00094381-05 0.8220 0.7147 0.86060.9614 0.8383 NCGC00015545-02 0.2480 0.2907 0.8475 0.9440 1.0179NCGC00164631-04 0.1827 0.2323 0.4405 0.6842 0.8225 NCGC00094112-040.3627 0.4403 1.0782 1.0309 1.1779 1.1721 NCGC00242500-01 0.4237 0.35702.4909 1.6977 1.1446 1.1021 NCGC00015545-07 0.3383 0.5257 0.8603 0.78961.1950 1.2967

TABLE 16 CMV-LACZ in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uMMLS000763074-01 1.9682 1.7671 0.9645 1.0487 0.8593 1.0089MLS000564846-01 1.0094 1.2012 0.8711 1.1234 1.0444 0.8304MLS001202366-01 1.0847 0.8329 0.9927 0.8072 1.0570 0.9844MLS001182278-01 1.1212 1.0224 1.0297 1.0327 1.0126 0.9674MLS001196427-01 0.9000 0.9918 0.9282 0.8423 1.0704 0.9363MLS001202425-01 0.9976 0.9918 1.0423 1.1496 1.0185 0.8793MLS001214276-01 0.4976 0.5259 11.1000 2.0151 0.9141 0.7733MLS000682748-01 1.1071 1.1753 0.9205 0.8790 0.8459 0.9356MLS000834756-01 2.2953 2.0788 1.1713 1.1994 1.0059 0.9859MLS001202402-01 0.8129 0.8847 0.8015 0.9237 0.8695 0.9200MLS001214264-01 1.4482 1.2424 1.1534 0.9411 1.1905 1.1210MLS000391588-01 0.9094 0.9212 0.9740 1.0019 1.0010 0.8400MLS001013431-01 1.2059 1.0682 0.9162 0.9388 1.1257 0.8638MLS001163848-01 6.8624 6.7729 4.2586 1.1937 0.9638 1.2133MLS000327715-01 1.4871 1.2200 0.9983 1.1289 1.0562 1.1762MLS001214300-01 0.7118 6.2635 1.0956 1.0550 0.8924 0.9819MLS000834755-01 0.8789 1.2989 1.1048 1.0514 1.2863 0.5547MLS001163860-01 1.4490 1.3880 1.2757 1.1250 1.1562 1.0352MLS000710233-01 1.1380 1.1130 1.0698 0.9060 0.9410 0.8781MLS002702497-01 0.6550 0.6520 6.3598 7.0726 2.2829 1.1181MLS000546982-01 1.3290 1.0160 1.1265 1.0159 0.8467 1.0438MLS001197838-01 1.0063 0.8547 1.1637 0.9701 1.0038 1.0305MLS001202354-01 0.7863 0.7000 1.0340 0.8910 0.9200 1.0676MLS001202330-01 0.8242 0.9168 0.8717 0.9600 0.9562 0.8314MLS001182377-01 1.0263 1.0305 1.0589 0.9608 0.8105 0.7762MLS000834757-01 1.3600 1.0789 1.1047 1.0546 0.8695 1.0238MLS001202634-01 0.9340 0.9750 0.9661 1.2348 1.0073 0.9582MLS001194544-01 0.9400 0.9800 0.9739 0.9683 1.1318 1.0764MLS000554109-01 0.9950 0.9100 1.1355 1.1559 0.9491 1.2682NCGC00263072-01 1.0230 0.9190 1.0660 1.0396 0.8536 1.0945NCGC00263071-01 0.9160 1.0840 0.9779 1.0165 1.2909 0.9845NCGC00263039-01 1.0510 0.8580 0.9982 0.9481 0.9727 0.9645NCGC00164631-03 0.7680 0.6690 0.3964 0.8420 1.2736 1.2682NCGC00179302-02 1.1410 1.0030 0.9196 1.1533 0.9736 0.9618NCGC00241113-01 0.9520 0.9790 1.0724 0.9410 1.3227 0.8627NCGC00094381-04 8.1220 3.8710 1.1010 1.2849 1.4927 1.0845NCGC00015546-04 1.0700 1.0230 1.1513 1.0553 1.1427 1.1555NCGC00094381-03 18.6030 10.9800 1.5444 1.1125 1.2227 0.9227NCGC00094381-05 18.0610 6.8950 2.8305 1.2069 0.8773 1.0045NCGC00015545-02 0.4770 0.5490 0.5058 0.7586 0.9536 0.8627NCGC00164631-04 0.6690 0.6490 0.3804 0.8803 0.9164 1.1336NCGC00094112-04 0.5420 0.7190 0.7933 0.8123 0.8864 1.0409NCGC00242500-01 7.0800 5.2430 3.3039 2.2432 1.9682 1.2482NCGC00015545-07 0.5470 0.7010 0.8393 0.9434 1.1036 1.0736

TABLE 17 407 compounds chosen from the initial 1500 MLSMR “hit”compounds and the results from the Burkin lab rescreening in myoblasts(MB), myotubes (MT), and β-Gal stabilizing assay Both MB and β-Gal Lessthan 25% 407 compounds MB >25% MT >25% MT >25% Stabilizing max increasein chosen for Burkin increase increase over increase over or CMV both MBand Lab evaluation over DMSO DMSO DMSO activating MT NCGC repeatedly 52(31%) 12 (7.2%) 10 (6%) 2* (1.2%) 90 (54%) active (166) Original tophits 6 (3%) 37 (18.8%) 12 (6.1%) 6** (3%) 136 (69%) (197) SU9516platform 6 (13.6%) 1 (2.3%) 3 (6.8%) 12*** (27.3%) 15 (34%) Analogs (44)*Neither of the β-Gal stabilizing compounds gave a >25% in α7^(+/LacZ)MB or MT assays **1 β-gal stabilizer also increased MT >25% over DMSO***1 β-gal stabilizer also increased MB >25% over DMSO

Top Hits from the Screen

Next the top 6 compounds for activation of ITGA7 in myotubes, based onmaximum response (Table 18) were selected and the screen was performedwith a larger number of concentrations in order to achieve more accuratedose-response curves (FIGS. 7A-7F). From these dose-response curves,GraphPad Prism nonlinear-regression analysis was used to determine theEC₅₀ in myotubes (Table 18). The maximum predicted increase wascalculated as the maximum increase generated in the α7^(+/LacZ)multiplied by 2 due to the single allelic copy of the LacZ reporter(FIGS. 7A-7F).

TABLE 18 Summary of the top 6 compounds found to be effective inmyotubes. Compound Known Online Online Screen EC50 MT FOLD Name MWeffects Screen Score Fold-change α7^(+/LacZ)MT INCREASE SU9516 241.3Cdk2 6.0 × 10⁻⁶M 2.4 inhibitor MLS000532969 236.3 NA 44 1.7 2.2 × 10⁻⁶M2.0 MLS003126425 399.4 NA 43 1.9 7.0 × 10⁻⁶M 2.0 MLS001060533 483.3 NA42 1.9 4.7 × 10⁻⁶M 1.5 MLS000683232 257.2 NA 5.3 × 10⁻⁶M 1.7MLS000683234 237.3 NA 46 2.6 2.7 × 10⁻⁵M 1.7SU9516 Increases α7 Integrin Protein in C2C12 Myotubes

In order to confirm the on-target activity of SU9516, C2C12 myotubeswere treated with either DMSO control or 12 μM SU9516 for 48 hours.Western blot analysis of the myotube protein extracts were thenperformed for α7B Integrin and normalized to GAPDH (FIGS. 8A and 8B).SU9516 treated myotubes displayed an increase of approximately 1.6-foldin α7 Integrin protein levels compared to DMSO treated controls (FIGS.8A and 8B). These results confirm the myogenic cell-based assay hadsuccessful identified novel small molecules that would target anincrease in α7 integrin in skeletal muscle.

Duchenne muscular dystrophy is a fatal neuromuscular disease for whichthere is currently no cure and limited treatment options. Studies haveshown that the α7β1 integrin is a major modifier of disease progressionin mouse models of DMD. Loss of the α7 integrin in dystrophin deficientmdx mice results in more severe muscle disease, while transgenic orviral mediated expression of the α7 integrin can rescue mouse models ofDMD. These results support the hypothesis that the α7β1 integrin ismajor modifier of disease progression in DMD. In support of the ideathat drug-based modulation of α7β1 integrin in skeletal muscle may serveas a therapeutic avenue for DMD, it has been shown that prednisone, thecurrent front line therapy for DMD, acts to increase laminin-α2 and α7β1integrin in the muscles of mdx mice, GRMD dog and DMD patient cells.Together these preclinical studies indicate the α7 integrin is a“druggable” target for the treatment of DMD.

In this study a muscle cell-based assay was used to identify smallmolecules that promote an increase in α7 integrin in myogenic cells. Ascreen of FDA-approved and novel compound libraries identified severalchemical platforms which may be promising for the treatment of DMD. Fromthe FDA-approved libraries it was identified that the Fe-chelatingcompounds Ciclopirox and Deferoxamine as α7β1 integrin enhancers. Inaddition 2,2-dipyridyl which is within this same compound family wastested using the muscle cell-based assay and was confirmed to alsoincrease α7 integrin levels. Since Ciclopirox, deferoxamine and2,2-dipyridyl have been shown to increase stability of the transcriptionfactor hypoxia inducible factor-1 (HIF-1) by preventing its breakdown,bioinformatic analysis was performed on the α7 integrin promoter.Analysis of a 2.8 kb fragment of the proximal α7 integrin promotersequence using MATINSPECTOR (Genomatix) revealed the presence of a HIF-1binding site and flanking sequences that promote HIF-1 binding. Theseresults indicate Ciclopirox, Deferoxamine and 2,2-dipyridyl act toincrease α7 integrin gene expression by inhibiting proteosomal breakdownof HIF-1 in muscle cells resulting in increased cellular levels of HIF-1protein in skeletal muscle. Interestingly, increased HIF-1 levels areassociated with increased angiogenesis and therefore not only wouldthese drugs increase membrane stability through increased α7 integringene expression, but they may also increase muscle vascularization,improving blood flow and reducing the ischemia associated withdystrophic muscle.

To identify small novel molecular probes that increase the α7 integrinin skeletal muscle, the MLSMR at the National Chemical and GenomicsCenter (NCGC) at NIH was screened. Using the muscle cell-based assay380,000 compounds in the Molecular Library and Small Molecule Repository(MLSMR) were screened using dose-response quantitative high through-putscreening (qHTS). From the primary screen, 1,500 hit compounds wereidentified as actives. Analysis of these compounds using thehierarchical cluster approach revealed 321 clusters in which 17 clusterscontained more than 10 compounds and 210 singletons. From these studies500 compounds were cherry-picked for further testing which includedconfirmation of activity in the primary screening assay and secondaryassays which included β-galactosidase reporter stabilizer, myostatin andcell-toxicity assays. In addition primary HTS active compounds weretested to determine if they increased β-galactosidase reporter inmyoblasts and myotubes. From these counter-screens seven novel hitcompounds were selected for further analysis to determine whether theyincrease the α7 integrin in a dose-dependent fashion. Analysis in mouseand DMD human muscle cells revealed that all seven increased both α7integrin transcript and protein levels. These compounds gave a maximalincrease in α7 integrin protein in human DMD muscle cells which would bein the therapeutic range as predicted from transgenic mouse studies.

Studies of the seven novel α7 integrin enhancing compounds showed thattwo molecular platforms are related, while the other compounds areunrelated to each other. One compound, SU9156 which was used as apositive control in the screen is currently in clinical trials as apotential anti-cancer therapeutic. SU9516(3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one) is a 3-substituted-indolinone compound that bindsto cdk2 and selectively inhibits its catalytic kinase activity. X-raycrystallography studies showed that the small molecule compoundinhibited cdk2/cyclin A through competitive inhibition of ATP. In humanleukemic cells, SU9516 caused the pronounced down-regulation of theanti-apoptotic protein Mcl-1 through transcriptional repression,increased proteasomal degradation, inhibition of RNA Pol II CTDphosphorylation and oxidative damage. Thus, SU9516 has demonstrated itspotential as a viable pharmacological drug for the development ofanti-neoplastic therapeutics and has reached clinical trials for thesame. The molecular mechanism by which SU9516 enhances integrin iscurrently unknown. SU9516 has also been reported to inhibit glycogensynthase kinase 3β (GSK-3β), which is involved in normal cell death.Levels of inactive p-S9-GSK3β are reduced and total GSK3β is elevated inthe muscles of patients with myotonic dystrophy type 1 (DM1). Inhibitionof GSK3β in both DM1 cell culture and mouse models reduced muscleweakness and myotonia in DM1 mice. Hence, compounds normalizing GSK3βactivity might be beneficial for improving muscle function in patientswith DM1.

Using a novel muscle cell-based assay an exhaustive screen of smallmolecule compound libraries has been conducted and identified severalFDA-approved and novel molecules that increase α7 integrin in skeletalmuscle. These small molecules can serve as molecular probes to dissectthe signaling pathways that regulate levels of the α7β1 integrin inskeletal muscle. In addition these molecules may serve as platforms todevelop novel therapeutics that target an increase in α7 integrin forthe treatment of muscular dystrophy.

Example 4

This example describes methods for identifying and analyzing suitableanalog compounds useful for the methods and assays disclosed herein. Inone embodiment, Stryka-969 was identified as a top “hit” from of theover 400,000 compounds screened using the assay disclosed herein due toits efficacy, large range of potency, and its lack of cellular toxicity(REF paper in prep). The chemical structure of Stryka-969 along with thedose-response curve generated in ITGA7^(+/Lacz) myotubes is illustratedFIG. 7B. While Stryka-969 also functions equally well in myoblasts, thetherapeutic target for enhanced α7 integrin protein are differentiatedmyofibers and thus this example focuses primarily on the Stryka-969analogs function in ITGA7^(+/Lacz) myotubes. The maximum increase of˜2-fold should translate into a 3-fold change in ITGA7 transcription dueto the heterogenetic nature of the assay and single reporter. Thisincrease is well within the therapeutic range previously reported fordystrophic mice.

FIG. 10 illustrates results obtained from some analogs of Stryka-969.These compounds were scored based on their structural similarity toStryka-969 using the Tanimoto coefficient which measures the similaritybetween two. These compounds were then used to treat ITGA7^(+/Lacz)myotubes at eight different concentrations in order to producedose-response curves (FIG. 10; n=3/concentration). The dose-responseresults suggest that the amide group may help increase α7 Integrin. Thisgroup is maintained in MLS000061149, which displays similar activity inthe Burkin assay to the parent compound Stryka-969 (FIG. 10). The othercompounds were less similar to Stryka-969 and displayed little to norelevant response (FIG. 10). Together, these studies indicate that SARanalysis will further elucidate the functional groups capable ofmaximizing the α7 integrin enhancing activity of this compound platform.

To assess the on-target effects of Stryka-969, culturedtelomerase-expressing Human DMD myotubes were used. After a minimum of10 days of differentiation, myotubes were treated with either DMSO orvarying concentrations of Stryka-969. After 48 hours the media wasaspirated and the myotubes were washed in PBS and then scraped intoice-cold RIPA buffer with protease inhibitors. After proteinconcentration was determined by BCA, standard SDS-PAGE and westernblotting procedures for α7B Integrin and α-Tubulin were followed. Amaximum increase of ˜1.7-fold over DMSO levels with a treatment of 20 μMStryka-969 (FIG. 11) was obtained. This shows that Stryka-969 is an α7integrin enhancing compound with strong therapeutic potential for thetreatment of DMD patients.

Pre-clinical assessment of therapeutic small molecules, including ADMETstudies, can help to prioritize small molecules with the highesttherapeutic value. A step to ensuring that small molecules will betherapeutically on-target in patients is to use cultured Human patientcells for assessment. This ensures that given biologic availability hasa conserved mechanism of action between species and the treatment shouldeffectively produce the desired effect in the specific tissue.

Briefly, either 1 mg or 5 mg of each analog are suspended in DMSO at 10μM concentration. Working plates are made to give a minimum of 8-pointdose response per compound. Drugs are added to ITGA7^(+/Lacz) myotubesfor 48 hours, at which time the FDG assay is performed as previouslydescribed. Using such analogs of Stryka-969 that achieve a minimum of1.5-fold increase in the Burkin Lab ITGA7^(+/Lacz) based assay, 6 cmplates with cultured Human DMD myotubes are treated with a range ofconcentrations based on the dose-response discussed above(n=3/concentration). After 48 hours the myotubes are washed and lysedfor western blot analysis. Several proteins, including α7B and β1AIntegrins, Laminin-α2, and utrophin levels, are assessed using standardwestern blotting techniques. If compounds fail to produce a minimum of1.5-fold α7 integrin increase they may be eliminated from furtherstudies. The behavior of Stryka-969 on the cultured Human DMD myotubesdiscussed herein indicates that analogs of this compound may lead toincreased α7 integrin in the Human cells. Western blots using a broadrange of concentrations can be used to avoid missing an optimal dose fora small molecule, which could occur if the EC₁₀₀ concentration from theinitial assay for use in the human DMD myotubes is solely relied upon.

The activity of other α7 integrin compounds also can be determined usingβ-galactosidase cleavage of the non-fluorescent compound fluoresceindi-β-D-galactopyranoside (FDG) to fluorescein in both myoblasts andmyotubes. For myoblast assays using analog compounds, 1×10⁴ cells areplated on 96 well black well culture plates and cells grown for 24hours. The parent and analog compounds are added and the FDG bioassay isperformed 48 hours after the addition of compounds. For myotube assays,2.5×10⁵ myoblasts on 96 well black cell culture plates are grown for 24hours. Cell differentiation medium is added daily and after 7 days ofdifferentiation, and compounds are added to myotubes and incubated for48 hours. β-galactosidase levels are then be quantified using the FDGassay. These assays re performed in triplicate for each compoundscreened. For each assay the parent compound and analogs are added tocells with a dose range from 0.5-20 μM in DMSO and a DMSO only controlis added for each assay. The dose curve class and EC₅₀ for each compoundis calculated, plotted and compared to the profile of the parent hitmolecule. Analog molecules that show activity (both positive andnegative) in myoblasts and myotubes inform the SAR for each molecularplatform series. In parallel, microsome and plasma stability,permeability and solubility of confirmed lead compounds able to increaseα7 integrin in myotubes is also evaluated to help determine the besttemplates for further pharmacokinetic evaluation. Lead compounds areselected for further studies based on the most potent activity inmyotubes (the target tissue) and optimal physical and structuraldrug-like properties in accordance with Lipinski's Rules.

Positive analogs are then assessed to determine whether they increase α7integrin transcript and protein using mouse C2C12 and human DMD myogeniccells. For myotube analysis, C2C12 and DMD myoblasts are differentiatedin DMEM supplemented with 2% horse serum and 50 U/ml ofpenicillin/streptomycin. Compounds or DMSO are added to C2C12 and DMDmyoblasts and myotubes at EC₁₀₀ dose calculated from the FDG screenabove. All studies are done in triplicate, and RNA is extracted usingTriZol reagent. RNA is reverse transcribed to cDNA using a SuperscriptII kit (Invitrogen). Primer sequences are used that specifically amplifymouse or human transcripts using SYBR Green technology and quantitativeRT-PCR, and reactions carried out in an ABI Prism 7000 SequenceDetection System. The C_(T) value and a standard curve from a dilutionseries of cDNA from non-treated cells is calculated by the accompanyingABI Prism 7000 SDS software. Transcripts are normalized to 18S rRNAtranscript and reported as fold change from control cells. Experimentsare performed in triplicate and statistical significance (p<0.05)determined using ANOVA.

Immunoblot analysis is used to assess the protein level expression intreated and control cells. C2C12 and human DMD myoblasts and myotubesare cultured in triplicate experiments and treated with lead compoundsor DMSO as described above. Cells are harvested using a cell scraper andextracted proteins. Extracted protein is quantified by a Bradford assay.Equal amounts of total protein are separated on SDS-PAGE gels at 40 mAfor 1 hour and protein is transferred to nitrocellulose membranes. Blotsare incubated with Ponceau S to confirm equal protein loading. Rabbitanti-α7 integrin antibodies, A2-345 and B2-347, at 1:500 with will beused to detect mouse and human α7A and α7B integrin respectively. Bandsare scanned with an Odyssey Infrared Imaging System. Blots are re-probedwith an anti-GAPDH to normalize band intensities for protein loading. ALiCoR Odyssey scanner and software is used to quantify band intensitiesand statistical significance (p<0.05) is determined using ANOVA.

The pharmacokinetic properties of the compounds having desirabledrug-like structures are analyzed for in vivo activity. Three 8-week-oldC57Bl/6 mice by will be treated by intraperiontoneal injection (i.p)with either the control solute or the compounds under investigation atthe EC₁₀₀ determined in the examples discussed above. At 0 mins (beforeadministration) and then 30 mins, 1 hr, 2 hrs, 4 hrs, 8 hrs, 12 hrs and24 hrs after compound administration, 50 μl of blood is collected byretro-orbital bleeds and serum isolated by centrifugation. Afteradministration, animals re observed and signs of toxicity includingweight loss, convulsions, uncoordinated movement, torpidity, temperaturechanges, dispenia or death. In a separate series of pharmacokineticexperiments to assess muscle tissue levels of analogs, three 8-week-oldC57Bl/6 mice are treated control solute or the compounds at the sametime points listed above. At each time point mice are humanelyeuthanized by CO2 inhalation and the gastrocnemius, TA, heart, intestineand diaphragm muscles dissected for analysis. The muscle and intestineis extracted to determine the levels of each lead analog within thetissue.

Purified compounds serve as a control to identify the small moleculesignature after LC-MS/MS analysis. Pharmacokinetic profiles include:serum half-life (t_(1/2)), Volume of distribution (V_(d)), drugconcentration in serum (C_(o) or C_(SS)), Elimination rate constant(k_(e)), Clearance (CL), Bioavailability (f), peak serum concentration(C_(max)) and time to reach C_(max) (t_(max)). A lead compound with asuitable pharmacokinetic profile is then selected for further studies.Formulation is developed with the objective of developing a lead α7integrin enhancing compound that can be orally administered. Forexperimental rigor, samples will be coded before analysis so thosecollecting data are blinded to the treatment group.

The pharmacokinetic profile for the lead compound identified above andthe calculated optimal dose are used to determine if the on-target invivo activity of the drug increases α7 integrin into the therapeuticrange as determined by transgenic mouse studies. Off-target activity ofthe lead compound also will be examined. Three week-old mdx mice areadministered with a suitable lead compound as determined using the aboveexamples, or a vehicle by oral gavage daily for 14 days using dosingdetermined in pharmacokinetic studies. C57Bl/10 mice are included aswild-type controls. A minimum of 22 male mdx mice per experimental andcontrol group are used as determined by Power analysis (Power=0.8,α=0.05 and r=0.5). Mice are weighed weekly and behavioral changesrecorded. Mice are euthanized at 5 weeks-of-age and Tibialis anterior(TA), gastrocnemius, diaphragm and cardiac muscles harvested. Expressionof α7 and β1 integrin is quantified by qRT-PCR and western blots asdiscussed herein. For off-target activity, transcript levels of integrinα3, α5, α6 and extracellular matrix genes are quantified. Primers areused that specifically amplify these mouse transcripts using SYBR Greentechnology and quantitative RT-PCR reactions performed in an ABI Prism7000 Sequence Detection System. The C_(T) value for each is calculatedusing ABI Prism 7000 SDS software and transcripts normalized to 18S rRNAand reported as fold change from control tissue. Experiments areperformed in triplicate and statistical significance (p<0.05) determinedusing ANOVA. Transcripts that change >2-fold are confirmed by westernblot analysis.

To determine if the lead compound shows therapeutic benefit, apreliminary muscle histology study also is performed. Before harvestingtissue, mice are injected with Evans Blue dye (EBD) and histologyperformed for EBD uptake, percentage of myofibers with centrally locatednuclei, inflammation, myofiber cross-sectional area and fibrosis aspreviously described. For experimental rigor, samples are coded beforeanalysis so those collecting data will blinded to the treatment group.Data analyzed by ANOVA and a p-value<0.05 will be consideredstatistically significant.

These examples can be used to determine the PK/PD of a lead α7 integrinenhancing compound. The lead compound with favorable PK/PD is thenassessed for on-target efficacy and off-target activity.

STATEMENTS OF THE DISCLOSURE

Paragraph 1. A method for treating a subject with muscular dystrophy,comprising administering an effective amount of an α7β1 integrinmodulatory agent to the subject with muscular dystrophy, wherein theα7β1 integrin modulatory agent is selected from any one or more ofFormulas 1-16 and/or any one of or more of the compounds disclosed inTables 1-16 and 18 and wherein the α7β1 integrin modulatory agentincreases α7β1 integrin expression or activity as compared to α7β1integrin expression or activity prior to treatment, thereby treating thesubject with muscular dystrophy.

Paragraph 2. The method disclosed in the preceding paragraph, whereinthe muscular dystrophy is merosin deficient congenital musculardystrophy Type 1A (MDC1A), merosin deficient congenital musculardystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy (LGMD),Duchenne muscular dystrophy (DMD), Fukuyama congenital musculardystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD).

Paragraph 3. The method disclosed in any one of the preceding paragraphswherein the muscular dystrophy is DMD, MDC1A or FCMD.

Paragraph 4. The method disclosed in any one of the precedingparagraphs, wherein the muscular dystrophy is DMD.

Paragraph 5. The method disclosed in any one of the precedingparagraphs, wherein the α7β1 integrin modulatory agent is administeredwith an additional therapeutic agent.

Paragraph 6. The method disclosed in the preceding paragraph, whereinthe additional therapeutic agent is a costameric protein, a growthfactor, satellite cells, stem cells, myocytes or an additional α7β1integrin modulatory agent.

Paragraph 7. The method disclosed in paragraph 6, wherein the additionalα7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment,valproic acid, or a valproic acid analog, a different compound selectedfrom Formulas 1-16 and/or Tables 1-16 and 18.

Paragraph 8. The method disclosed in any one of the precedingparagraphs, further comprising selecting a subject with musculardystrophy.

Paragraph 9. The method disclosed in the preceding paragraphs, whereinselecting a subject with muscular dystrophy comprises diagnosing thesubject with muscular dystrophy prior to administering an effectiveamount of the α7β1 integrin modulatory agent to the subject.

Paragraph 10. A method of enhancing muscle regeneration, repair, ormaintenance in a subject, comprising:

administering an effective amount of an α7β1 integrin modulatory agentto the subject in need of muscle regeneration, repair, or maintenance,wherein the α7β1 integrin modulatory agent is selected from any one ormore of Formulas 1-16 and/or any one of or more of the compoundsdisclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatoryagent increases α7β1 integrin expression or activity as compared to α7β1integrin expression or activity prior to treatment, thereby enhancingmuscle regeneration, repair or maintenance in a subject.

Paragraph 11. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of the preceding paragraph, wherein the α7β1modulatory agent is administered prior to the subject experiencingmuscle damage or disease.

Paragraph 12. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of any one of the preceding paragraphs, whereinthe method is a method of enhancing muscle maintenance in a subject.

Paragraph 13. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of any one of the preceding paragraphs, whereinthe α7β1 integrin modulatory agent is administered to the subject priorto the subject exercising.

Paragraph 14. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of any one of the preceding paragraphs, whereinthe α7β1 integrin modulatory agent is administered to a subject at riskof acquiring a muscle disease or damage.

Paragraph 15. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of any one of the preceding paragraphs, furthercomprising selecting a subject in need of enhancing muscle regeneration,repair, or maintenance.

Paragraph 16. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of the preceding paragraph, wherein selecting asubject in need of enhancing muscle regeneration, repair, or maintenancecomprises diagnosing the subject with a condition characterized byimpaired muscle regeneration prior to administering an effective amountof the α7β1 integrin modulatory agent to the subject.

Paragraph 17. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of any one of the preceding paragraphs, whereinselecting a subject in need of enhancing muscle regeneration, repair, ormaintenance comprises diagnosing the subject with a conditioncharacterized by impaired production of a component of α7β1 integrinprior to administering an effective amount of the α7β1 integrinmodulatory agent to the subject.

Paragraph 18. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of any one of the preceding paragraphs, whereinthe α7β1 integrin modulatory agent is administered with an additionaltherapeutic agent.

Paragraph 19. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of the preceding paragraph, wherein theadditional therapeutic agent is a costameric protein, a growth factor,satellite cells, stem cells, myocytes or an additional α7β1 integrinmodulatory agent.

Paragraph 20. The method of enhancing muscle regeneration, repair, ormaintenance in a subject of the preceding paragraph, wherein theadditional α7β1 integrin modulatory agent is laminin-111, a laminin-111fragment, valproic acid, or a valproic acid analog, or a differentcompound selected from Formulas 1-16 and/or Tables 1-16 and 18.

Paragraph 21. A method of prospectively preventing or reducing muscleinjury or damage in a subject, comprising, administering an effectiveamount of an α7β1 integrin modulatory agent to the subject wherein theα7β1 integrin modulatory agent is selected from any one or more ofFormulas 1-16 and/or any one or more of the compounds disclosed inTables 1-16 and 18 and wherein the α7β1 integrin modulatory agentincreases α7β1 integrin expression or activity as compared to α7β1integrin expression or activity prior to treatment, therebyprospectively preventing or reducing muscle injury or damage in thesubject.

Paragraph 22. The method of the preceding paragraph, wherein the subjectis at risk of developing a muscle injury or damage.

Paragraph 23. The method of prospectively preventing or reducing muscleinjury or damage in a subject in any one of the preceding paragraphs,wherein the α7β1 integrin modulatory agent is administered with anadditional therapeutic agent.

Paragraph 24. The method of prospectively preventing or reducing muscleinjury or damage in a subject of the preceding paragraph, wherein theadditional therapeutic agent is a costameric protein, a growth factor,satellite cells, stem cells, myocytes or an additional α7β1 integrinmodulatory agent.

Paragraph 25. The method of prospectively preventing or reducing muscleinjury or damage in a subject of the preceding paragraph, wherein theadditional α7β1 integrin modulatory agent is laminin-111, a laminin-111fragment, valproic acid, or a valproic acid analog, or a differentcompound selected from Formulas 1-16 and/or Tables 1-16 and 18.

Paragraph 26. A method of enhancing α7β1 integrin expression, comprisingcontacting a cell with an effective amount of an α7β1 integrinmodulatory agent, wherein the α7β1 integrin modulatory agent is selectedfrom any one or more of Formulas 1-16 and/or any one or more of thecompounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrinmodulatory agent increases α7β1 integrin expression in the treated cellrelative to α7β1 integrin expression in an untreated cell, therebyenhancing α7β1 integrin expression.

Paragraph 27. The method of the preceding paragraph, wherein the cell isa muscle cell.

Paragraph 28. The method of enhancing α7β1 integrin expression of any ofthe preceding paragraphs, wherein the muscle cell is present in amammal, and wherein contacting the cell with an agent comprisesadministering the agent to the mammal.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method of enhancing muscle regeneration, repair, ormaintenance in a subject, comprising: administering an effective amountof an α7β1 integrin modulatory agent to the subject in need of muscleregeneration, repair, or maintenance, wherein the α7β1 integrinmodulatory agent is

and wherein the α7β1 integrin modulatory agent increases α7β1 integrinexpression or activity as compared to α7β1 integrin expression oractivity prior to treatment, thereby enhancing muscle regeneration,repair or maintenance in a subject.
 2. The method of claim 1, whereinthe α7β1 integrin modulatory agent is administered prior to the subjectexperiencing muscle damage or disease.
 3. The method of claim 1, whereinthe method is a method of enhancing muscle maintenance in a subject. 4.The method of claim 3, wherein the α7β1 integrin modulatory agent isadministered to the subject prior to the subject exercising.
 5. Themethod of claim 3, wherein the α7β1 integrin modulatory agent isadministered to a subject at risk of acquiring a muscle disease ordamage.
 6. The method of claim 1, further comprising selecting a subjectin need of enhancing muscle regeneration, repair, or maintenance.
 7. Themethod of claim 6, wherein selecting a subject in need of enhancingmuscle regeneration, repair, or maintenance comprises diagnosing thesubject with a condition characterized by impaired muscle regenerationprior to administering an effective amount of the α7β1 integrinmodulatory agent to the subject.
 8. The method of claim 6, whereinselecting a subject in need of enhancing muscle regeneration, repair, ormaintenance comprises diagnosing the subject with a conditioncharacterized by impaired production of a component of α7β1 integrinprior to administering an effective amount of the α7β1 integrinmodulatory agent to the subject.
 9. The method of claim 1, wherein theα7β1 integrin modulatory agent is administered with an additionaltherapeutic agent.
 10. The method of claim 9, wherein the additionaltherapeutic agent is a costameric protein, a growth factor, satellitecells, stem cells, myocytes or an additional α7β1 integrin modulatoryagent.
 11. The method of claim 10, wherein the additional α7β1 integrinmodulatory agent is laminin-111, a laminin-111 fragment, valproic acid,or a valproic acid analog.
 12. The method of claim 1, wherein the methodis a method of enhancing muscle regeneration.
 13. The method of claim12, wherein the α7β1 integrin modulatory agent is administered to asubject with a muscle disease or damage.
 14. The method of claim 13,wherein the muscle disease is a muscular dystrophy.
 15. The method ofclaim 14, wherein the muscular dystrophy is merosin deficient congenitalmuscular dystrophy Type 1A (MDC1A), merosin deficient congenitalmuscular dystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy(LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital musculardystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD). 16.The method of claim 15, wherein the muscular dystrophy is DMD, MDC1A orFCMD.
 17. The method of claim 15, wherein the muscular dystrophy is DMD.